Temperature dependent probes for target detection

Abstract

The present invention provides oligonucleotides and methods for their use in the detection and / or differentiation of one or more targets in a sample. In some examples, the oligonucleotides and methods find particular application in amplifying, detecting, and / or discriminating multiple targets simultaneously.

Description

[0001] TEMPERATURE DEPENDENT PROBES FOR TARGET DETECTION

[0002] Technical Field

[0003] The present invention relates generally to the field of molecular biology. More specifically, the present invention provides oligonucleotides and methods for their use in the detection and / or differentiation of targets. The oligonucleotides and methods find particular application in amplifying, detecting, discriminating and / or quantifying multiple targets simultaneously.

[0004] Incorporation by Reference

[0005] The present application claims priority from Australian provisional application number 2024903744 filed on 14 November 2024, the entire contents of which are incorporated herein by reference.

[0006] Background

[0007] Genetic analysis is routinely used in the clinic for assessing disease risk, diagnosis of disease, predicting a patient's prognosis or response to therapy, and for monitoring a patient's progress. The introduction of such genetic tests depends on the development of simple, inexpensive, and rapid assays for discriminating genetic variations.

[0008] Methods of in vitro nucleic acid amplification have wide-spread applications in genetics and disease diagnosis. Such methods include polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), strand displacement amplification (SDA), nicking enzyme amplification reaction (NEAR), helicase dependent amplification (HDA), Recombinase Polymerase Amplification (RPA), loop-mediated isothermal amplification (LAMP), rolling circle amplification (RCA), transcription-mediated amplification (TMA), self-sustained sequence replication (3 SR), nucleic acid sequence based amplification (NASBA), Ligase Chain Reaction (LCR) or Ramification Amplification Method (RAM). Most of these target amplification strategies requires the use of oligonucleotide primer(s). In most protocols, the process of amplification results in the accumulation of amplicons which incorporate the oligonucleotide primers at their 5 ’ termini of each strand, and which contain newly synthesised copies of the sequences located between the primers.

[0009] One method for monitoring the accumulation of amplicons in real-time, or at the conclusion of amplification, involves detection using PlexZymes, also known in the literature as Multi-component Nucleic Acid Enzymes or MNAzymes. PlexZymes have been previously described to cleave probes which are dual-labelled universal substrates which have either a linear or a hairpin conformation. Linear substrates were the first type of reporter probes described for MNAzymes / PlexZymes and may be considered as “standard” substrates or probes. Hairpinned PlexZyme substrates are known in the art as either LOCS (Loops Connected to Stems) Probes or PlexPlus Probes. Recently, a novel type of PlexZyme substrate was developed, known as M-Tec (Multi-component Temperature-Controlled) probes (Australian Provisional Patent Application No.

[0010] 2023901468). LOCS and M-Tec probes have specific features which allow control of signal generation via manipulation of temperature. Other methods for monitoring the accumulation of amplicons in real-time, or at the conclusion of amplification, include detection target-specific Molecular Beacons, Sloppy Beacons, Binary DNA probes (also known as universal Molecular Beacons), Eclipse probes, TaqMan Probes or Hydrolysis probes, Scorpion Uni-Probes or Bi-Probes, Catcher / Pitcher probes for TOCE technology, Dual Hybridisation probes, Double-stranded probes (Yin-Yang probes) and / or the use of intercalating dyes such as SYBR Green.

[0011] Melt curve analysis can be performed during or at the conclusion of several of these protocols to obtain additional information since amplicons with different sequences denature at different temperatures, known as the melting temperature or Tm. Such protocols measure melting curves which result from either a) the separation of the two strands of double stranded amplicons in the presence of an intercalating dye, or b) the separation of one strand of the amplicon and a complementary target-specific probe labelled with a fluorophore and quencher or c) separation of non-target related duplexes, for example, Catcher duplexes which are only generated in the presence of target. Melt curve analysis provides information about the dissociation kinetics of two DNA strands during heating. The melting temperature (Tm) is the temperature at which 50% of the DNA is dissociated. The Tm is dependent on the length, sequence composition and G-C content of the paired nucleotides. Elucidation of information about the target DNA from melt curve analysis conventionally involves a series of fluorescence measurements acquired at small temperature intervals, typically over a broad temperature range. In some protocols the derivative of this curve is then plotted as a function of temperature to obtain the melt curve. Melting temperature does not only depend upon on the sequence of the nucleic acid strands. The Tm can be influenced by many factors including the concentrations of oligonucleotides, cations in the buffer (both monovalent (Na+) and divalent (Mg2+) salts), and / or the presence or absence of destabilising agents such as urea or formamide. Melt curve analysis protocols are often slow and typically take between 30-60 mins to complete. Furthermore, melt curve analyses can require interpretation by skilled personnel and / or the use of specialised software for results interpretation. Hence, there is a high demand for faster and / or less complex alternatives to melt curve analyses. Further, melt curves are typically analysed post-PCR and therefore only allow for a qualitative determination of the presence or absence of target in a sample. In many instances, a quantitative, or semi -quantitative, determination of the amount of genomic material present in a sample is required. Therefore, there is a high demand for fast alternatives to melt curve analysis that also provide quantitative information about a sample.

[0012] Hairpin probes or Stem-Loop probes have proven to be useful tools for detection of nucleic acids and / or monitoring target amplification. One type of hairpin probe, which is dual labelled with a fluorophore and quencher dye pair, is commonly known in the art as a Molecular Beacon. In general, these molecules have three features; 1) a Stem structure formed by hybridisation of complementary 5' and 3' ends of the oligonucleotide; 2) a loop region which is complementary to the target, or target amplicon, to be detected; and 3) a fluorophore quencher dye pair attached at the termini of the Molecular Beacon. During PCR, the loop region binds to the amplicons due to complementarity and this causes the stem to open thus separating the fluorophore quencher dye pair. An essential feature of Molecular Beacons is that the loop regions of these molecules remain intact during amplification and are neither degraded or cleaved in the presence of target or target amplicons. The separation of the dye pair attached on the termini of an open Molecular Beacon causes a change in fluorescence which is indicative of the presence of target to which it is hybridised. The method is commonly used for multiplex analysis of multiple targets in a single PCR test. In general, for multiplex analysis, each Molecular Beacon has a different target-specific loop region and a unique fluorophore, such that hybridisation of each different Molecular Beacons to each amplicon species can be monitored in a separate channel i.e. at a separate wavelength. A disadvantage of Molecular Beacons is that careful design and reaction temperature optimisation is required to balance the transition between the hairpin conformation and the linear conformation adopted when the Molecular Beacons binds to the target.

[0013] The concept of Molecular Beacons has been extended in a strategy known as Sloppy Beacons. In this protocol the loop region of a single Beacon is long enough such that it can tolerate mismatched bases and hence bind to a number of closely related targets differing by one or more nucleotides. Following amplification, melt curve analysis is performed and different target species can be differentiated based on the temperature at which a separation (melt) of each of the duplexes formed by hybridisation of the target species with the loop region of a Sloppy Beacon occurs. In this way multiple closely related species can be detected at a single wavelength and discriminated simultaneously by characterising the melting profile of specific targets with the single Sloppy Beacon. Standard Molecular Beacons and Sloppy Beacons differ from TaqMan and Hydrolysis probes in that they are not intended to be degraded or cleaved during amplification. A disadvantage of DNA hybridisation-based technologies such as sloppy beacons and TOCE is that they may produce false positive results due to non-specific hybridisation between probes and nontarget nucleic acid sequences.

[0014] In general, the number of available fluorescent channels capable of monitoring discrete wavelengths limits the number of targets which can be detected, and specifically identified, in a single reaction on a fluorescent reader. Recently, two approaches have been described which allow detection and quantification of two or more targets at a single wavelength. The first approach utilises post-amplification analysis (i.e. amplification curve analysis = ACA) to enable quantitative detection of multiple targets at a single wavelength. The second approach relies on generation of independent fluorescence signals at a single wavelength, eliminating the need for ACA.

[0015] Use of ACA allows use of several different types of fluorescent qPCR probes; two example protocols are described below in detail. In addition to these protocols, ChromaCode strategy requires manipulation of the data from a single temperature to extract the desired data for individual targets (see, e.g., International Patent Publication No. WO / 2017 / 173035). The first protocol, known as “Tagging Oligonucleotide Cleavage and Extension” (TOCE), uses Pitcher and Catcher oligonucleotides. Pitchers have two regions, the Targeting Portion, which is complementary to the target, and the Tagging portion which is non-complementary and located at the 5' terminus. The Catcher oligonucleotide is dual labelled and has a region at its 3 ' end which is complementary to the tagging portion of the Pitcher. During amplification, the Pitcher binds to the amplicons and when the primers extend the 5 '-3' exonuclease activity of the polymerase can cleave the Tagging portion from the Pitcher. The released Tagging portion then binds to the Catcher Oligonucleotide and functions as a primer to synthesise a complementary strand. The melting temperature of the double stranded Catcher molecule (Catcher-Tm) then acts as a surrogate marker for the original template. Since it is possible to incorporate multiple Catchers with different sequences and lengths, all of which melt at different temperatures, it is possible to obtain a series of Catcher-Tm values indicative of a series of targets whilst still measuring at a single wavelength. Limitations with this approach include inherent complexity as it requires the released fragment to initiate and complete a second extension on an artificial target, and post amplification analysis of multiple targets requires complex algorithms to differentiate or quantify the proportion of signal related to each specific target. The method measures fluorescence at various temperatures, however at the lowest temperature all double stranded Catcher molecules fluoresce giving a combined signal for all targets. At the highest temperature where florescence is acquired, only one double stranded Catcher molecule, which has the highest Tm, will remain double stranded and hence will fluoresce. An algorithm is then required to determine the contribution from the one or more targets detected in the reaction.

[0016] In the second protocol requiring ACA, multiple LOCS reporters, can be combined in the same reaction to measure multiple targets at a single wavelength. Intact LOCS reporters or probes contain a stem region, labelled with a fluorophore quencher dye pair at each terminus, and a Loop region which comprises a substrate for an enzyme. In the presence of a target, the substrate can be cleaved or hydrolysed resulting in Split LOCS structures. Enzymes suitable for mediating target-dependent substrate modification include catalytic nucleic acids such as an PlexZymes and DNAzymes, or protein enzymes such as exonucleases or endonucleases. The melting temperatures of the stem regions of Intact LOCS (Intact LOCS Tm) are higher than those of resultant Split LOCS (Split LOCS Tm) since intramolecular bonds are stronger than intermolecular bonds. Both the Intact LOCS and the Split LOCS will be either quenched, or will generate fluorescence, depending upon whether the temperature of the reaction milieu is above or below the melting temperature of their stems. The presence of fluorescence at temperatures below the Intact LOCS Tm but above the Split LOCS Tm is indicative of the presence of the target which facilitates the cleavage. The target can be directly detected, or target amplicons produced by target amplification protocols, can be detected. Multiple LOCS reporters can be combined to facilitate detection of multiple targets at a single wavelength in a single reaction. In such reactions all LOCS are labelled with dyes that can be monitored at the same wavelength; however, each has a stem designed to melt at a different temperature, and each has a loop that is cleavable only in the presence of its specific target. In real time PCR, fluorescence specific for two LOCS / two targets can be acquired at two temperatures where a lower temperature is set to be suitable for measuring fluorescence associated with the Split LOCS with the lower Tm stem only. A second higher temperature is suitable for measuring the combined signal for both high and low Tm Split LOCS but will not generate fluorescence from either LOCS probe when Intact.

[0017] Recently two protocols have been described which allow detection of multiple targets at a single wavelength in real time without the need for an ACA to determine the contribution from the one or more targets. The common feature to these protocols is utilisation of probes that generate temperature-specific, independent fluorescence signals. The first protocol combines LOCS reporters with other probe types, for example Molecular Beacons in single reactions where probe pairs are labelled with the fluorophores that produce fluorescence in the same channel. At the first detection temperature, the Molecular Beacon can bind to a first target, causing spatial separation of the fluorophore and quencher and an increase in fluorescence. At a second higher detection temperature, at which it can neither bind to its target, nor form a quenched hairpin, the Molecular Beacon can adopt a random coil structure, which causes only constant level of background fluorescence regardless of the presence or absence of the first target. The LOCS reporter is designed to be cleaved in the presence of a second target and to have a Split LOCS Tm that is above the first detection temperature but below the second detection temperature. As such, at the first temperature the LOCS reporter remains quenched regardless of the presence of absence of the second target but causes increased fluorescence at the higher temperature if there has been target dependent cleavage of Intact LOCS. Overall, the Molecular Beacon is fluorescent at the first lower detection temperature if the first target is present, and the LOCS is quenched regardless of the presence or absence of the second target; whilst at the second, higher detection temperature the Molecular Beacon generates only background fluorescent at a constant level, which is unaffected by the presence or absence of the first target and the LOCS is fluorescent only if the second target is present. As such each temperature measures changes in florescence associated with only one type of probe in the presence of a target and hence no algorithm is required to ascertain a signal specific for each target and probe type. Whilst this protocol is advantageous and flexible, it uses Molecular Beacons or other probe types which often have restrictive design requirements with respect to temperature; and / or may involve the use of specific reagents including, for example, polymerases which lack exonuclease activity.

[0018] To overcome limitations related to Molecular Beacons, LOCS probes were recently combined with a novel universal probe called M-Tec (PCT / AU2024 / 050265). M-Tec Probes are multiple-component complexes composed of at least two oligonucleotide components wherein a first oligonucleotide component (OC1) is capable of being modified by an enzyme only in the presence of a specific target and a second oligonucleotide component (OC2) is labelled with a second detection moiety. The first oligonucleotide component comprises a first capture region capable of hybridisation to the second oligonucleotide component by complementary base pairing to form a double-stranded portion. The first and second oligonucleotide components are capable of hybridisation at temperatures below the melting temperature (Tm) of the double-stranded portion (Tm OC1 / OC2). When all oligonucleotide components are hybridised, and the first oligonucleotide component is unmodified, the fluorophore and quencher are in close proximity resulting in intact M-Tec Probe complexes which are quenched. In the presence of a target, a sensor region of the first oligonucleotide component is modified, for example by cleavage or hydrolysis by an enzyme. Similarly to LOCS, enzymes suitable for mediating target-dependent substrate modification include catalytic nucleic acids such as an PlexZymes and DNAzymes, or protein enzymes such as exonucleases or endonucleases.

[0019] Enzymatic modification of the sensor region of the first oligonucleotide component generates a first fragment comprising the first capture region and a second fragment connected to the first detection moiety, thereby enabling the first and second detection moi eties to spatially separate and generate a first detectable signal. These target-dependent increases in fluorescence can be measured at temperatures below Tm OC1 / OC2. At temperatures above the Tm OC1 / OC2, the first and second oligonucleotide components dissociate, and the dye moieties are separated, resulting in background levels of florescence which are constant regardless of the presence or absence of target.

[0020] The combination of LOCS and M-Tec or Molecular Beacon probe allow generation of two temperature-specific, independent fluorescence signals at a single fluorescent wavelength; additional detection temperatures i.e. more than two detection temperatures at a single channel, however, require use of ACA due to the thermodynamic capabilities of these probes. Both LOCS and M-Tec probes are fully universal i.e. they can be used to detect any target.

[0021] The second protocol that allows independent temperature-specific fluorescent signals is based on PTOCE method. This protocol describes four alternative probe compositions that allow detection of two targets at a single wavelength in real time without the need for ACA (WO 2022 / 265463 Al). The first probe composition consists of two PTOCE method based probes: second composition consists of a PTOCE method based probe and dualquenched probe; third composition consists of two PTOCE method based probes; fourth composition consists of a PTOCE method based probe and dual -quenched probe.

[0022] An extensive description of PTOCE (Probing and Tagging Oligonucleotide Cleavage and Extension) method can be found elsewhere (WO 2012 / 096523) and has similarities with previously described TOCE method. In short, PTOCE-based methods encompass various methods for providing signals, comprising of the formation of an extended strand through cleavage and extension of a PTO. Probing and tagging oligonucleotide (PTO) comprises of a 3 ’targeting portion complementary to the target sequence and a 5 ’-tagging portion non-complementary to the target sequence. A capturing and templating oligonucleotide (CTO) comprises of a capturing portion complementary to the 5 ’-tagging portion of a part of the 5 ’-tagging portion of the PTO and a templating portion non-complementary to the 5’-tagging portion and 3’-targeting portion of the PTO. The common signal generation method by PTOCE comprises of the following steps: (a) induced cleavage of the PTO by an enzyme having 5’ nuclease activity, in a way that releases a fragment comprising of the 5’-tagging portion of the PTO; (b) hybridising the fragment released from the PTO with a CTO; (c) extension of the fragment hybridised to the capturing portion of the CTO to form an extended duplex. The extended duplex has a Tm value that can be adjusted by the sequence length; it has at least one label linked to the fragment and / or the CTO and is capable of providing a fluorescent signal at a predetermined temperature. The dual labels may be linked to CTO only, or one label may be linked to CTO and one to PTO; other modifications also exist. Depending on where and how the labels are attached, signal generation mechanism can be altered to make the probe suitable for different detection temperatures. WO 2012 / 096523 gives examples of PTOCE-based probes suitable for different detection temperatures (first, second and third). It should be noted that PTOCE probes may be either universal or non -universal; whenever there’s a label attached to the target-specific PTO, the probe is not considered universal. PTOCE probes consisting of unlabelled PTOs are considered universal.

[0023] The dual-quenching method, originally combined with melting analysis (WO 2016 / 101959), involves formation of a duplex by cleavage of a mediation oligonucleotide. It involves a mediation oligonucleotide linked to a reporter molecule and first quencher molecule (also referred to as a PTO), and a capture oligonucleotide (also referred to as a Capturing and Quenching Oligonucleotide (CQO)) linked to a second quencher molecule. The PTO and CQO are designed to form a duplex, and the cleavage of PTO is dependent on the presence of target. PTO oligonucleotide comprises of (1) a targeting portion complementary to the target nucleic acid sequence, (ii) a nucleotide sequence non-complementary to the target nucleic acid also called Melting Temperature Deciding Region (MTDR) and (iii) at least one set of interactive labels comprising at least one fluorophore and at least one quencher. CQO comprises of (i) a capturing portion comprising a nucleotide sequence reverse complementary to the MTDR of the mediator oligonucleotide, (ii) at least of quenching molecule. Contacting the probe duplex with an enzyme having nuclease activity induces cleavage of the PTO, releasing the activated PTO / CQO duplex comprising of a fragment of PTO hybridised to CQO. This PTO / CQO duplex which remains hybridised i.e. quenched under temperature below Tm of the said duplex, and is dissociated i.e. unquenched at temperatures above Tm of the duplex. It should be noted that dual-quenching method is not considered a universal probe as it involves a labelled target-specific PTO.

[0024] In addition to the four probe compositions for two detection temperatures, WO 2022 / 265463 Al also describes a composition of three different probes that allow independent fluorescence signals at three detection temperatures at a single wavelength. This composition consists of two PTOCE-method based probes and dual-quenched probe. The first nucleic acid target is detected by PTOCE-based method where all interactive dual labels are linked to a CTO; the second nucleic acid target is detected by PTOCE-method where one of the interactive labels is linked to a PTO and the other to the CTO; the third nucleic acid target is detected by dual -quenching method. The first PTOCE-based probe is designed to form a PTO / CTO duplex that is in a dissociated form at all three temperatures when target is not present; when target is present, the extended duplex is formed at the first detection temperature only and is in a dissociated form at second and third detection temperatures. This allows generation of fluorescence at first detection temperature only as an indication of presence of target. Second PTOCE-based probe consists of a PTO having a quencher molecule linked to its tagging portion, and a CTO having a reporter molecule linked to its capturing portion; when uncleaved, they were designed to form a duplex at the first detection temperature and a dissociated form at second and third detection temperatures. When target is present and PTO gets cleaved, the extended duplex is formed at first and second detection temperature and is dissociated at third detection temperature, which results in extinction of fluorescence at the second detection temperature as an indication of presence of target. The last dual-quenched probe consists of a PTO linked to a reporter molecule and a first quencher molecule, and a CQO linked to a second quencher molecule; in the absence of target, they were designed to form a taq duplex at the first and second detection temperatures and to dissociate at the third detection temperature. In the presence of target, PTO is cleaved and generates an activated taq duplex which is in associated form at the first and second detection temperatures, and in dissociated form at the third detection temperature. This allows independent generation of fluorescence at the third temperature. It should be noted that only one of the probes involved in this system is a fully universal probe; both the second PTOCE probe and dual -quenched probe involve a labelled target-specific PTO. Therefore, this probe combination has same limitations as other target-specific probes including high assay development costs; a new probe has to be designed for each new target, which makes development of new assays more complex and time-consuming. Furthermore, all PTOCE methods require in vitro amplification wherein primers amplify and the 5 ’-3’ exonuclease activity of the DNA polymerases cleaves the PTO. Other probe technologies relying on in vitro amplification include TaqMan or other hydrolysis probes.

[0025] A need exists for improved compositions and methods for the simultaneous detection, differentiation, and / or quantification of multiple unique amplicons generated by PCR or by alternative target amplification protocols. It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.

[0026] Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

[0027] Summary of the Invention

[0028] Provided herein are methods and compositions which extend the capacity for multiplex analysis of nucleic acid targets. These methods and compositions employ T emperature-dependent Looped Multi-component Probes, herein referred to as TLM Probes. By way of example, TLM Probes are multiple-component complexes composed of at least two oligonucleotide components wherein a first oligonucleotide (Oligo 1) is capable of being modified by an enzyme only in the presence of a specific target. The key feature of this probe is that the effective binding affinity between probe components is different in intact and cleaved probes. The effective binding affinity of the probe is lowered once the Oligo 1 is cleaved by enzymatic modification in the presence of target. This in turn lowers the effective melting temperature (Tm) of the cleaved probe i.e. temperature that allows full separation of the fluorophore from the quencher. This means that an uncleaved probe has a higher effective Tm compared to an intact probe. Therefore, discrimination between presence and absence of target can be detected / measured at temperatures above Tmcleavedand below Tmintactt. On the other hand, at temperatures below Tmcleavedboth intact and cleaved probes are non-fluorescent (quenched). At temperatures above Tmintact, both intact and cleaved probes fluoresce. Therefore, at temperatures above Tmintact and below Tmcleaved, intact and cleaved probes produce the same fluorescence level. In summary, the probe is capable of producing a target-dependent signal at a confined temperature range.

[0029] Within the probe structure, Oligo 1 comprises at least two capture regions. Oligo 1 capture region 1 capable of hybridisation to capture region 1 of the second oligonucleotide (Oligo 2) by complementary base pairing to form a double-stranded portion. Oligo 1 also comprises of a sensor region that is capable of being modified by enzymatic activity. Oligo 1 is connected to a detection moiety by direct labelling or indirect labelling, for example via a third region of complementarity with a third oligonucleotide component (Oligo 3) which is directly labelled with the detection moiety. Thus, where oligonucleotide 1 is indirectly labelled, there may be additional sequence between the label and Oligo 1. Oligo 2 is also labelled with a second detection moiety. This labelling may be direct or indirect labelling. Where oligonucleotide 2 is indirectly labelled, there may be additional sequence between the label and the oligonucleotide. The first and second detection moiety may be, for example, a fluorophore and a quencher or vice versa. As mentioned above, Oligo 1 also comprises capture region 2. The sensor region lies between capture region 1 and 2. Depending on which other region this capture region is complementary to, two distinct exemplary structures can be distinguished. These are described in the next two paragraphs.

[0030] The first exemplary structure (Figure 1) comprises capture region 2 complementary to capture region 3 in Oligo 1. When the capture region 2 and 3 are hybridised, an internal stem-loop structure is formed in Oligo 1. The sensor region lies between capture regions 2 and 3. The binding affinity between the capture regions 2 and 3 depends on whether the Oligo 1 is intact or cleaved (Figure 1 (i) and (ii)). In intact probes, the hybridised capture regions 2 and 3 form an internal stem-loop structure; in cleaved probes, the two capture regions are separated into different Oligo 1 fragments. Since intramolecular bonds are stronger than intermolecular bonds, the Tm of the stem-loop structure in intact probes is higher than the Tm of capture region of the cleaved probes i.e. the Tm of the capture region 2 and 3 in Oligo 1 is different in intact and cleaved probes. In a cleaved probe, separation of fluorophore from the quencher requires Oligo 1 capture region 2 / 3 only to dissociate, therefore the effective Tmcleavedis Tm Oligo 1 capture region 2 / 3 only. In an intact probe, separation of fluorophore from the quencher requires dissociation of capture region 1 between Oligo 1 and Oligo 2 only; therefore, Tmintact is Tm capture region 1. In an embodiment, in an intact probe, Tm capture region 1 is higher than Tm capture region 2 / 3. As the effective Tmintact is higher than Tmcleaved, a fluorescence signal is generated at temperatures above Tmcleavedand below Tmintact in the presence of target only.

[0031] The second exemplary structure (Figure 2) comprises a second complementary region between Oligo 1 and Oligo 2. This second double-stranded region is formed by the second capture region of Oligo 1 hybridising to the second capture region of the Oligo 2 by complementary base pairing. Therefore, there are two complementary regions between Oligo 1 and Oligo 2 and enzymatic cleavage of Oligo 1 occurs between the two regions. When the probe is intact, both of these complementary regions need to dissociate for full separation of fluorophore from the quencher, therefore the binding affinity in both regions contributes towards the effective Tm between Oligo 1 and Oligo 2 (Figure 2 (i) and (ii)).

[0032] The first complementary region increases the Tm of the second complementary region and vice versa, due to the increased binding affinity between Oligo 1 and Oligo 2 when there are two complementary regions between them. This means that the effective Tmintact for this type of probe is combined Tm of the capture regions. In a cleaved probe, the two capture regions in Oligo 1 are separated into the separate fragments; the Tm of the complementary regions is now independent from each other, and the effective Tmcleavedis Tm complementary region 2 only as only the second Oligo 1 fragment containing capture region 2 needs to dissociate from the probe complex to allow separation of the fluorophore and quencher. In an embodiment, Tm capture region 1 is higher than Tm capture region 2. As the effective Tmintact is higher than Tmcleaved, a signal is generated at temperatures above Tmcieaved and below Tmintact in the presence of target only.

[0033] As indicated above, both intact and cleaved TLM probes of both described structures can exist in completely bound, quenched form or fully dissociated, fluorescent form. At temperatures below effective Tmcleaved, the TLM probe is always in a bound and quenched form due to close proximity of the fluorophore and quencher, regardless of whether the probe is intact (target not present) or cleaved (target present). On the other hand, at temperatures above effective Tmintact, the TLM probe is always fully dissociated and fully fluorescent due to full separation of the fluorophore from the quencher, resulting in background fluorescence regardless presence or absence of target. In this manner TLM Probes will generate target dependent increases in fluorescence only at temperatures above effective Tmcleavedand below effective Tmintact but no change in fluorescence will be observed regardless of the presence or absence of target at temperatures below effective Tmcieaved and above the effective Tmintact.

[0034] If present, a third oligonucleotide component (Oligo 3) is capable of hybridisation with the Oligo 1 via complementary capture regions in Oligo 1 and Oligo 3. The Tm of this capture region should be above effective Tmintact, to ensure that the Oligo 1 and Oligo 3 remain hybridised at temperatures below effective Tmintact..

[0035] Various types of TLM Probes are disclosed and exemplified. One type of TLM Probe is suitable for modification / cleavage by an PlexZyme (i.e., an MNAzyme). These probes, denoted herein as TLM-P probes, have a sensor region in the Oligo 1 which can serve as a substrate for a PlexZyme. In some embodiments, the Oligo 2 hybridises to a capture region of the Oligo 1 which does not hybridise / bind to the substrate binding arms of the PlexZyme. The TLM-P probe can bind to the substrate binding arms of a PlexZyme when one assembles in the presence of its specific target. Cleavage of the sensor region of the TLM-P Probe results in generation of a first fragment and a second fragment of Oligo 1. Following the same principle as described above, TLM-P probe generates target dependent increases in fluorescence only at temperatures above Tmcleavedand below Tmintact but no change in fluorescence will be observed regardless of the presence or absence of target at temperatures below Tmcleavedand above the Tmintact- Example of this probe is provided in Figures 3, 4 and 5. This type of TLM is fully universal, similar to LOCS and M-Tec probes. Another type of TLM Probe is suitable for cleavage by exonuclease activity, for example, 5 '-3' exonuclease of Taq polymerase, in the presence of target. In these probes, denoted herein as TLM-H probes, the sensor region of the Oligo 1 includes a sequence which is complementary to the target to be detected. In some embodiments the Oligo 2 hybridises to a region of the Oligo 1 which does not hybridise / bind to the target. During PCR, the Oligo 1 binds to the target amplicons and is hydrolysed by the exonuclease activity of polymerase, resulting in generation of a first fragment and a second fragment. Following the same principle as described above, TLM-H probe generates target dependent increases in fluorescence only at temperatures above Tmcleavedand below Tmintact but no change in fluorescence will be observed regardless of the presence or absence of target at temperatures below Tmcleavedand above the Tmintact- Another type of TLM Probe is suitable for cleavage by endonuclease activity, for example, a nicking endonuclease in the presence of target. In these probes, denoted herein as TLM-E probes, the sensor region of the Oligo 1 includes a sequence which is complementary to the target to be detected. In some embodiments the Oligo 2 hybridises to a region of the Oligo 1 which does not hybridise / bind to the target. The Oligo 1 of the TLM-E probe can bind to the target and form a double stranded recognition site for a nicking enzyme. This enzyme can then cleave the Oligo 1, resulting in generation of a first fragment and a second fragment while leaving the target intact. In some embodiments, a first fragment generated from cleavage of the Oligo 1 by the endonuclease retains the capacity to form the first double-stranded portion by hybridisation to Oligo 2. Following the same principle as described above, TLM-H probe generates target dependent increases in fluorescence only at temperatures above Tmcleavedand below Tmintact but no change in fluorescence will be observed regardless of the presence or absence of target at temperatures below Tmcleavedand above the Tmintact. In other scenarios, TLM probe can be cleaved by a restriction enzyme which cleaves both strands of a duplex.

[0036] In some embodiments, TLM probes are suitable for use in methods where there is direct detection of the target i.e. in reactions that are not subjected to in vitro amplification. In other embodiments, TLM probes are suitable for use in conjunction with in vitro amplification. Isothermal in vitro amplification protocols may utilise any MNAzyme or endonuclease compatible with the reaction temperature. When in vitro amplification involves thermocycling, for example PCR, thermostable endonucleases may be preferred.

[0037] In some embodiments, TLM Probes are used in combination with LOCS Probes and M-Tec probes. In other embodiments, M-Tec Probes can be combined with other probe and substrate types well known in the art which include, but are not limited to, dual labelled linear PlexZyme substrates, TaqMan probes or Hydrolysis probes, Molecular Beacons, Binary DNA probes (universal Molecular Beacons), Sloppy Beacons, Eclipse probes, Scorpion Uni-Probes or Bi-Probes, Capture / Pitcher Oligonucleotides, Double-stranded probes (Yin-Yang probes) and dual-hybridisation probes. In other embodiments, multiple TLM Probes can be used in combination with each other.

[0038] The combination of multiplex TLM Probes and TLM Probe with other probe or substrate types allows greater multiplexing capacity, wherein multiple targets may be detected, identified and / or qualified at a single wavelength. By way of example, an TLM Probe, together with a LOCS probe and M-Tec probe, both of which incorporate the same detection moiety (e.g. the same fluorophore) may be used to individually discriminate multiple targets within a single reaction. The approach involves measurement of the signal generated from the probes at discrete temperatures. In some embodiments a first target is measured at a first temperature by monitoring any changes in fluorescence associated with modification of an M-Tec Probe, second target is measured at a second temperature by monitoring any changes in fluorescence associated with modification of a TLM probe and a third target is measured at a third temperature by monitoring any changes in fluorescence associated with modification of a LOCS probe. In another example, two TLM probes together with a LOCS probe and M-Tec probe, all of which incorporate the same detection moiety (e.g. the same fluorophore) may be used to individually discriminate four targets within a single reaction. In some embodiments a first target is measured at a first temperature by monitoring any changes in fluorescence associated with modification of an M-Tec Probe, second target is measured at a second temperature by monitoring any changes in fluorescence associated with modification of a first TLM probe, third target is measured at a third temperature by monitoring any changes in fluorescence associated with modification of a second TLM probe and a fourth target is measured at a fourth temperature by monitoring any changes in fluorescence associated with modification of a LOCS probe

[0039] The present disclosure relates to at least the following embodiments:

[0040] 1. A method for determining the presence or absence of a target in a sample, the method

[0041] comprising:

[0042] (a) preparing a mixture for a reaction by contacting the sample or a derivative thereof putatively comprising the target with:

[0043] - a temperature-dependent looped multi-component probe (TLM) comprising a first oligonucleotide component and a second oligonucleotide component, wherein the first oligonucleotide component comprises a first capture region and a second capture region, the first capture region being capable of hybridisation to a first capture region of the second oligonucleotide component by complementary base pairing to form a first double-stranded portion, and the second capture region being capable of hybridisation to a third capture region of the first oligonucleotide component or to a second capture region of the second oligonucleotide component by complementary base pairing to form a second double-stranded portion,

[0044] wherein the first oligonucleotide component further comprises a single-stranded loop portion of unhybridised nucleotides comprising a sensor region capable of serving as a substrate for an enzyme, wherein the sensor region is located between the first and second capture regions of the first oligonucleotide component;

[0045] wherein the first oligonucleotide component is connected to a first detection moiety and the second oligonucleotide component is connected to a second detection moiety; wherein the first detection moiety of the first oligonucleotide is connected to the second capture region of the first oligonucleotide;

[0046] - an enzyme capable of modifying the sensor region of the first oligonucleotide component only when the target is present in the sample;

[0047] (b) treating the mixture under conditions suitable for the enzyme to modify the sensor region of the first oligonucleotide component to thereby generate a first fragment comprising the first capture region of the first oligonucleotide and a second fragment connected to the first detection moiety, thereby enabling the first and second detection moieties to spatially separate and generate a first detectable signal,

[0048] (c) measuring a level of background signal or detectable signal generated at a defined temperature at or below which the first capture region is hybridised to the second oligonucleotide component; and

[0049] (d) determining the presence or absence of the target based upon the level of detectable signal measured at the defined temperature, wherein a detectable signal at the defined temperature is indicative of the presence of the target in the sample.

[0050] 2. The method of embodiment 1 wherein the enzyme is capable of digesting the sensor region of the first oligonucleotide component only when the target is present in the sample, and wherein step (b) comprises treating the mixture under conditions suitable for the enzyme to digest the sensor region of the first oligonucleotide component to thereby generate a first fragment comprising the first capture region of the first oligonucleotide and a second fragment connected to the first detection moiety.

[0051] 3. The method of embodiment 1 or embodiment 2 wherein the method comprises: (i) measuring a level of background signal or detectable signal generated by the first and second detection moieties in the mixture at the defined temperature

[0052] - at a timepoint prior to or during said treating the mixture, and

[0053] - at one or more subsequent timepoint(s) during or following said treating the mixture; and (ii) determining a presence of or a change in the level of detectable signal which differs from the background signal and is indicative of the presence of the target in the sample.

[0054] 4. The method of embodiment 3 wherein step (c) comprises measuring the detectable signal or any said background signal:

[0055] - at one or more timepoints prior to said treating;

[0056] - at one or more timepoints during said treating;

[0057] - at one or more timepoints after said treating;

[0058] - at one or more timepoints during said treating and at one or more timepoints after said treating;

[0059] - at one or more timepoints prior to said treating and at one or more timepoints after said treating; or

[0060] - at one or more timepoints before and during said treating and at one or more timepoints after said treating.

[0061] 5. The method of embodiment 3 or embodiment 4 wherein step (d) comprises using a predetermined threshold value to determine if the detectable signal differs from any said background signal at the defined temperature.

[0062] 6. The method of embodiment 1 or embodiment 2 further comprising measuring a level of control background signal generated at the defined temperature in a control mix; wherein step (c) comprises measuring a level of the background or detectable signal in the mixture contacted by the sample or derivative thereof; and

[0063] wherein step (d) comprises determining whether a detectable signal that differs from the control background signal is generated and indicative of the presence of the target in the sample.

[0064] 7. The method of embodiment 1 or embodiment 2 further comprising:

[0065] measuring a level of control background signal generated at the defined temperature in a control mix, and

[0066] determining whether the level of control background signal measured in the control mix differs from the level of background signal or detectable signal measured in the mixture at step (c), wherein a difference in the level of background signal or detectable signal measured in the mixture at step (c) compared to the level of control background signal measured in the control mix is indicative of the presence of the target in the sample.

[0067] 8. The method of embodiment 6 or embodiment 7 wherein the control mix does not comprise the target but is otherwise equivalent to the mixture.

[0068] 9. The method of embodiment 6 or embodiment 7 wherein the control mix does not comprise the enzyme but is otherwise equivalent to the mixture.

[0069] 10. The method of embodiment 1 or embodiment 2 further comprising:

[0070] measuring a level of control detectable signal generated at the defined temperature in a control mix, wherein the control mix comprises a predetermined amount of the target but is otherwise equivalent to the mixture; and

[0071] determining whether the level of control detectable signal measured in the control mix differs from the level of background signal or detectable signal measured in the mixture at step (c),

[0072] wherein a difference in the level of background signal or detectable signal measured in the mixture at step (c) compared to the level of control detectable signal measured in the control mix is indicative of the presence and / or amount of the target in the sample.

[0073] 11. The method of any one of embodiments 1 to 10 wherein the target is a nucleic acid and at least a portion of the sensor region hybridises to a complementary sequence in the target to thereby form a duplex between the sensor region and the target.

[0074] 12. The method of embodiment 11 wherein the enzyme is an endonuclease that recognises a sequence in the duplex.

[0075] 13. The method of embodiment 12 wherein the endonuclease digests at least one strand of the duplex to thereby form the first and second fragments.

[0076] 14. The method of embodiment 12 wherein the endonuclease is a nicking endonuclease that digests the sensor region of the first oligonucleotide component after formation of the duplex to thereby form the first and second fragments.

[0077] 15. The method of embodiment 11 wherein the enzyme is an exonuclease that hydrolyses the sensor region of the first oligonucleotide component after formation of the duplex to thereby form the first and second fragments.

[0078] 16. The method of embodiment 15 wherein the exonuclease is a polymerase with exonuclease activity.

[0079] 17. The method of embodiment 16 wherein

[0080] - at least a portion of the sensor region hybridises to a complementary sequence in the target to thereby form a duplex between the sensor region and the target, - said mixture further comprises a target primer capable of binding to the target at a region upstream of said complementary sequence,

[0081] - said treating the mixture comprises:

[0082] hybridisation of the target primer to the target by complementary base pairing, extending the primer using the polymerase with exonuclease activity and using the nucleic acid target as a template,

[0083] wherein the polymerase comprising exonuclease activity digests the sensor region of the first oligonucleotide component after formation of the duplex to thereby form the first and second fragments.

[0084] 18. The method of any one of embodiments 1 to 10 wherein the enzyme is a DNAzyme.

[0085] 19. The method of any one of embodiments 1 to 10 wherein the target is a nucleic acid and the sensor region of the first oligonucleotide component is not complementary to the target.

[0086] 20. The method of any one of embodiments 1 to 10 or 19 wherein the target is a nucleic acid and the enzyme is a multi-component nucleic acid enzyme (MNAzyme) comprising two partzyme oligonucleotides capable of self-assembling to form the MNAzyme only in the presence of the target.

[0087] 21. The method of embodiment 20 wherein said treating comprises:

[0088] hybridising sensor arms of the MNAzyme to the target by complementary base pairing, and hybridising substrate arms of the MNAzyme to at least a portion of the sensor region of the first oligonucleotide component by complementary base pairing to facilitate cleavage of the first oligonucleotide component and generation of the first and second fragments. 22. The method of any one of embodiments 1 to 10 wherein the target is a nucleic acid. 23. The method of embodiment 22 wherein the target is an amplicon of a nucleic acid. 24. The method of embodiment 23 wherein the amplicon is produced by an amplification reaction selected from the group consisting of polymerase chain reaction (PCR), strand displacement amplification (SDA), nicking enzyme amplification reaction (NEAR), helicase dependent amplification (HD A), Recombinase Polymerase Amplification (RPA), loop-mediated isothermal amplification (LAMP), rolling circle amplification (RCA), transcription-mediated amplification (TMA), self-sustained sequence replication (3 SR), nucleic acid sequence based amplification (NASBA), Ligase Chain Reaction (LCR) or Ramification Amplification Method (RAM) and reverse transcription polymerase chain reaction (RT-PCR).

[0089] 25. The method of embodiment 24, wherein said detecting:

[0090] - occurs prior to said amplification or within 1, 2, 3, 4, or 5 cycles of said amplification commencing; and / or - occurs after completion of said amplification.

[0091] 26. The method of any one of embodiments 23 to 25 wherein said determining the presence or absence of the target comprises a melt curve analysis.

[0092] 27. The method of any one of embodiments 1 to 10 wherein:

[0093] - the enzyme is a DNAzyme or a ribozyme requiring a co-factor for catalytic activity, - said treating of the mixture comprises using conditions suitable for:

[0094] binding of the cofactor to the DNAzyme or ribozyme to render it catalytically active, hybridisation of the DNAzyme or ribozyme to the first oligonucleotide component by complementary base pairing,

[0095] catalytic activity of the DNAzyme or ribozyme to thereby digest the first oligonucleotide component and generate the first fragment and the second fragment, and

[0096] - the target is the co-factor.

[0097] 28. The method of embodiment 27 wherein the co-factor is a metal ion, such as a metal ion selected from: Mg2+, Mn2+, Ca2+ and Pb2+.

[0098] 29. The method of any one of embodiments 1 to 10 wherein the enzyme is an aptazyme wherein:

[0099] - the sensor region comprises a substrate for an aptazyme;

[0100] - the target is an analyte, protein, peptide, compound or nucleic acid;

[0101] - the mixture comprises an aptazyme comprising an aptamer capable of binding to the target; and

[0102] - said treating the mixture further comprises binding of the aptazyme to the target and to the sensor region to facilitate cleavage of the first oligonucleotide component to thereby generate the first fragment and the second fragment.

[0103] 30. A method for determining the presence or absence of a target in a sample, the method comprising:

[0104] (a) preparing a mixture for a reaction by contacting the sample or a derivative thereof putatively comprising the target with:

[0105] - a temperature-dependent looped multi-component probe (TLM) comprising a first oligonucleotide component and a second oligonucleotide component,

[0106] wherein the first oligonucleotide component comprises a first capture region and a second capture region, the first capture region being capable of hybridisation to a first capture region of the second oligonucleotide component by complementary base pairing to form a first double-stranded portion, and the second capture region being capable of hybridisation to a third capture region of the first oligonucleotide component or to a second capture region of the second oligonucleotide component by complementary base pairing to form a second double-stranded portion,

[0107] wherein the first oligonucleotide component further comprises a single-stranded loop portion of unhybridised nucleotides comprising a sensor region capable of serving as a substrate for an enzyme, wherein the sensor region is located between the first and second capture regions of the first oligonucleotide component,

[0108] wherein the first oligonucleotide component is connected to a first detection moiety and the second oligonucleotide component is connected to a second detection moiety; wherein the first detection moiety of the first oligonucleotide is connected to the second capture region of the first oligonucleotide;

[0109] - an enzyme capable of modifying the sensor region of the first oligonucleotide component only when the target is present in the sample;

[0110] (b) treating the mixture under conditions suitable for the enzyme to modify the sensor region of the first oligonucleotide component to thereby generate a first fragment comprising the first capture region of the first oligonucleotide and a second fragment connected to the first detection moiety, thereby enabling the first and second detection moieties to spatially separate and generate a first detectable signal,

[0111] (c) measuring a level of detectable signal generated at a defined temperature at or below which the first capture region hybridises to the second oligonucleotide component; and

[0112] (d) determining the presence or absence of the target based upon the presence or absence of a change in detectable signal generating a melt curve peak, wherein absence of a melt curve peak is indicative of the presence of the target in the sample and presence of a melt curve peak is indicative of the absence of the target in the sample.

[0113] 31. The method of any one of embodiments 1 to 30 wherein the second oligonucleotide component is directly labelled with the second detection moiety.

[0114] 32. The method of any one of embodiments 1 to 31 wherein the first fragment is not directly labelled with a detection moiety.

[0115] 33. The method of any one of embodiments 1 to 32 wherein the second fragment is not directly labelled with the first detection moiety.

[0116] 34. The method of any one of embodiments 1 to 33 wherein the first oligonucleotide component is not directly labelled with the first detection moiety.

[0117] 35. The method of any one of embodiments 1 to 34 wherein the second capture region of the first oligonucleotide component is capable of hybridisation to a third capture region of the first oligonucleotide component by complementary base pairing to form the second double-stranded portion.

[0118] 36. The method of any one of embodiments 1 to 34 wherein the first oligonucleotide further comprises a third capture region capable of hybridisation to the second capture region of the first oligonucleotide component by complementary base pairing to form the second double-stranded portion.

[0119] 37. The method of any one of embodiments 1 to 34 wherein the second capture region of the first oligonucleotide component is capable of hybridisation to a second capture region of the second oligonucleotide component by complementary base pairing to form the second double-stranded portion.

[0120] 38. The method of any one of embodiments 1 to 37 wherein the first double-stranded portion of the TLM probe has a melting temperature (Tm) that is above the defined temperature.

[0121] 39. The method of any one of embodiments 1 to 38 wherein the Tm of the second doublestranded portion is less than the Tm of the first double-stranded portion.

[0122] 40. The method of any one of embodiments 1 to 33 wherein the first oligonucleotide component is directly labelled with the first detection moiety.

[0123] 41. The method of any one of embodiments 1 to 40 wherein the TLM probe does not comprise more than two detection moieties.

[0124] 42. The method of any one of embodiments 1 to 41 wherein the first oligonucleotide component is not directly labelled with more than one detection moiety

[0125] 43. The method of any one of embodiments 1 to 41 wherein the first oligonucleotide component is not connected to more than one detection moiety.

[0126] 44. The method of any one of embodiments 1 to 43 wherein the second oligonucleotide component is not directly labelled with more than one detection moiety or is not connected to more than one detection moiety.

[0127] 45. The method of any one of embodiments 1 to 44 wherein:

[0128] the first detection moiety is a fluorophore, and the second detection moiety is a quencher; or

[0129] the first detection moiety is a quencher, and the second detection moiety is a fluorophore,

[0130] optionally wherein the TLM probe does not comprise more than one quencher or optionally wherein the detectable signal is fluorescence emitted in the presence of the target.

[0131] 46. The method of any one of embodiments 1 to 45 wherein neither the first oligonucleotide component or the second oligonucleotide component serve as a primer for a DNA polymerase in an extension reaction and / or wherein neither the first oligonucleotide component or the second oligonucleotide serve as a template for a DNA polymerase in an extension reaction.

[0132] 47. The method of any one of embodiments 1 to 46 wherein the presence or absence of the target in a sample is determined at temperatures above the melting temperature (Tm) of the cleaved TLM probe and below the Tm of the intact TLM probe.

[0133] 48. The method of any one of embodiments 1 to 47 wherein the second oligonucleotide component is not enzymatically cleaved or degraded.

[0134] 49. The method of any one of embodiments 1 to 48 wherein the sensor region is located between the first capture region of the first oligonucleotide component and the first detection moiety or between the second capture region of the first oligonucleotide component and the third capture region of the first oligonucleotide component.

[0135] 50. The method of any one of embodiments 1 to 49 wherein following said treating the mixture the first fragment is capable of hybridising to the second oligonucleotide component via the first capture region.

[0136] 51. The method of any one of embodiments 1 to 50 wherein the second capture region of the first oligonucleotide is capable of hybridisation to a third capture region of the first oligonucleotide component by complementary base pairing to form a second doublestranded portion, and the probe further comprises a third oligonucleotide component comprising a first capture region capable of hybridisation to a fourth capture region of the first oligonucleotide component by complementary base pairing to form a third doublestranded portion.

[0137] 52. The method of any one of embodiments 1 to 50 wherein the second capture region of the first oligonucleotide is capable of hybridisation to a second capture region of the second oligonucleotide component by complementary base pairing to form a second doublestranded portion, and the probe further comprises a third oligonucleotide component comprising a first capture region capable of hybridisation to a third capture region of the first oligonucleotide component by complementary base pairing to form a third doublestranded portion.

[0138] 53. The method of embodiment 51 or 52 wherein the third oligonucleotide is connected to a detection moiety or wherein the third oligonucleotide is directly labelled with a detection moiety.

[0139] 54. The method of embodiment 53 wherein the detection moiety is the first or the second detection moiety, optionally wherein the first detection moiety is a fluorophore, and the second detection moiety is a quencher; or the first detection moiety is a quencher, and the second detection moiety is a fluorophore.

[0140] 55. The method of any one of embodiments 1 to 54 wherein the biological sample is obtained from a subject and / or generation of the detectable signal at the defined temperature is not reversible.

[0141] 56. The method of any one of embodiments 1 to 55 wherein the method is performed in vitro or ex vivo.

[0142] 57. A method for determining the presence or absence of a first target and a second target in a sample, the method comprising:

[0143] (a) preparing a mixture for a reaction by contacting the sample or a derivative thereof putatively comprising the first and / or second target with:

[0144] - a temperature-dependent looped multi-component (TLM) probe for detection of the first target, the TLM probe comprising a first oligonucleotide component and a second oligonucleotide component;

[0145] wherein the first oligonucleotide component comprises a first capture region and a second capture region, the first capture region being capable of hybridisation to a first capture region of the second oligonucleotide component by complementary base pairing to form a first double-stranded portion, and the second capture region being capable of hybridisation to a third capture region of the first oligonucleotide component or to a second capture region of the second oligonucleotide component by complementary base pairing to form a second double-stranded portion;

[0146] wherein the first oligonucleotide component further comprises a single-stranded loop portion of unhybridised nucleotides comprising a sensor region capable of serving as a substrate for an enzyme, wherein the sensor region is located between the first and second capture regions of the first oligonucleotide component;

[0147] wherein the first oligonucleotide component is connected to a first detection moiety and the second oligonucleotide component is connected to a second detection moiety; wherein the first detection moiety of the first oligonucleotide is connected to the second capture region of the first oligonucleotide;

[0148] - a second nucleic acid probe for detection of the second target, the second nucleic acid probe comprising third and fourth detection moieties,

[0149] wherein the first and second detection moieties are capable of generating a first detectable signal, and the third and fourth detection moieties are capable of generating a second detectable signal, and - a first enzyme capable of modifying the sensor region of the first oligonucleotide component only when the first target is present in the sample;

[0150] (b) treating the mixture under conditions suitable for:

[0151] - the first enzyme to modify the sensor region of the first oligonucleotide component to thereby generate a first fragment comprising the first capture region of the first oligonucleotide and a second fragment connected to the first detection moiety, thereby enabling the first and second detection moieties to spatially separate and generate a first detectable signal,

[0152] - the second target to induce a modification of the second nucleic acid probe, thereby enabling the third and fourth detection moieties to spatially separate and generate a second detectable signal;

[0153] (c) measuring a level of background or detectable signal:

[0154] - at a first temperature at or below which the first capture region is hybridised to the second oligonucleotide component,

[0155] - at a second temperature at or above which the first capture region is not hybridised to the second oligonucleotide component,

[0156] (d) determining whether at one or more timepoints during or after said treating: - a first detectable signal is generated at the first temperature at or below which the first capture region is hybridised to the second oligonucleotide component,

[0157] - a second detectable signal arising from said modification of the second nucleic acid probe is generated at the second temperature, wherein the second detectable signal is indicative of the presence of the second target in the sample.

[0158] 58. The method of embodiment 57 wherein the first enzyme is capable of digesting the sensor region of the first oligonucleotide component only when the first target is present in the sample, and wherein step (b) comprises treating the mixture under conditions suitable for the first enzyme to digest the sensor region of the first oligonucleotide component to thereby generate a first fragment comprising the first capture region of the first oligonucleotide and a second fragment connected to the first detection moiety.

[0159] 59. The method of embodiment 57 or embodiment 58 wherein a first detectable signal at the first temperature is indicative of the presence of the first target in the sample.

[0160] 60. The method of embodiment 59 wherein the presence of the first target is determined at the first temperature based upon the first detectable signal generated at the first temperature.

[0161] 61. The method of any one of embodiments 57 to 60 wherein the presence of the second target is determined at the second temperature based upon the second detectable signal generated at the second temperature. 62. The method of embodiment 57 or embodiment 58 wherein:

[0162] (i) at the first temperature

[0163] - a first detectable signal is generated in the presence of the first target,

[0164] - a second detectable signal is generated in the presence of the second target, or

[0165] - a first detectable signal and a second detectable signal is generated in the presence of both the first target and the second target; and

[0166] (ii) a second detectable signal is generated at the second temperature only in the presence of the second target.

[0167] 63. The method of any one of embodiments 57 to 62 wherein the method comprises: - measuring a level of background signal or detectable signal at the first and second temperatures generated by the first and second detection moieties and by the third and fourth detection moieties in the mixture,

[0168] - determining a presence of or a change in the level of the first detectable signal which differs from the background signal and is indicative of the presence of the first target in the sample, and

[0169] - determining a presence of or a change in the level of the second detectable signal arising from said modification generated at the second temperature which differs from the background signal and is indicative of the presence of the second target in the sample. 64. The method of any one of embodiments 57 to 63 wherein at the second temperature, dissociation of the second oligonucleotide component from the capture region of either the first oligonucleotide component present in the absence of the first target, or the first fragment generated by modification of the first oligonucleotide component in the presence of the first target generate an equal, similar or equivalent background signal.

[0170] 65. The method of any one of embodiments 57 to 64 wherein step (c) comprises measuring a level of background signal at a third temperature and at the third temperature the second capture region remains hybridised in the presence or absence of the target, thereby generating a background signal.

[0171] 66. The method of any one of embodiments 57 to 65 wherein said determining comprises detection of the first detectable signal and / or any said background signal:

[0172] - at one or more timepoints prior to said treating;

[0173] - at one or more timepoints during said treating;

[0174] - at one or more timepoints after said treating;

[0175] - at one or more timepoints during said treating and at one or more timepoints after said treating; - at one or more timepoints prior to said treating and at one or more timepoints after said treating; or

[0176] - at one or more timepoints before and during said treating and at one or more timepoints after said treating.

[0177] 67. The method of any one of embodiments 57 to 66, wherein said determining in part (d) comprises:

[0178] - using a predetermined threshold value to determine if the first detectable signal differs from any said background signal at the first temperature; and / or

[0179] - using a predetermined threshold value to determine if the second detectable signal differs from any said background signal at the second temperature.

[0180] 68. The method of any one of embodiments 57 to 63 comprising:

[0181] measuring a level of first control background signal at the first temperature provided by the first and second detection moieties and by the third and fourth detection moieties in a control mix;

[0182] measuring a level of second control background signal at the second temperature provided by the first and second detection moieties and by the third and fourth detection moieties in the control mix;

[0183] determining whether a level of the first detectable signal generated at the first temperature at step (c) in the mixture contacted by the sample or derivative thereof differs from the level of first control background signal measured in the control mix, wherein a difference in the level of detectable signal measured in the mixture at the first temperature at step (c) compared to the first control background signal measured in the control mix is indicative of the first target in the sample; and

[0184] determining whether a level of the second detectable signal generated at the second temperature at step (c) in the mixture contacted by the sample or derivative thereof differs from the level of second control background signal measured in the control mix, wherein a difference in the level of detectable signal measured in the mixture at the second temperature at step (c) compared to the second control background signal measured in the control mix is indicative of the second target in the sample.

[0185] 69. The method of embodiment 68 wherein the control mix does not comprise:

[0186] - the first target;

[0187] - the second target; or

[0188] - the first and second targets,

[0189] but is otherwise equivalent to the mixture. 70. The method of embodiment 68 wherein the control mix does not comprise the first enzyme but is otherwise equivalent to the mixture.

[0190] 71. The method of any one of embodiments 57 to 63 further comprising:

[0191] measuring a level of first detectable signal generated at the first temperature in a control mix, wherein the control mix comprises a predetermined amount of

[0192] the first target,

[0193] the second target, or

[0194] the first and second targets,

[0195] but is otherwise equivalent to the mixture;

[0196] measuring a level of second detectable signal generated at the second temperature in the control mix;

[0197] determining whether a level of the first detectable signal generated at the first temperature at step (c) in the mixture contacted by the sample or derivative thereof differs from the level of first control detectable signal measured in the control mix, wherein a difference in the level of detectable signal measured in the mixture at the first temperature at step (c) compared to the first control detectable signal measured in the control mix is indicative of the first target in the sample; and

[0198] determining whether a level of the second detectable signal generated at the second temperature at step (c) in the mixture contacted by the sample or derivative thereof differs from the level of second control detectable signal measured in the control mix, wherein a difference in the level of detectable signal measured in the mixture at the second temperature at step (c) compared to the second control detectable signal measured in the control mix is indicative of the second target in the sample.

[0199] 72. The method of any one of embodiments 57 to 67 wherein part (c) comprises measuring a first background signal at or within 1°C, 2°C, 3°C, 4°C or 5°C of the first temperature, and a second background signal at or within 1°C, 2°C, 3°C, 4°C or 5°C of the second temperature.

[0200] 73. The method of embodiment 72 wherein part (d) comprises determining whether at one or more time points during or after said treating:

[0201] a first detectable signal is generated at the first temperature which differs from the first background signal and is indicative of the presence of the first target in the sample; and

[0202] a second detectable signal is generated at the second temperature which differs from the second background signal and is indicative of the presence of the second target in the sample. 74. The method of any one of embodiments 57 to 73 wherein at the first temperature the third and fourth detection moieties do not generate a signal which differs from the background signal.

[0203] 75. The method of any one of embodiments 57 to 74 wherein at the second temperature the first and second detection moieties do not generate a signal which differs from the background signal.

[0204] 76. The method of any one of embodiments 57 to 75 wherein the first and second detectable signals are detectable by a single detector optionally wherein the first and second detectable signals are detectable in the same fluorescent channel.

[0205] 77. The method of any one of embodiments 57 to 76 wherein the first and second detectable signals are detectable as fluorescent emission at a single wavelength.

[0206] 78. The method of any one of embodiments 57 to 77, wherein the first and second detection moieties, and the third and fourth detection moieties emit a detectable signal at the same or similar wavelength which can be detected in the same fluorescence channel.

[0207] 79. The method of any one of embodiments 57 to 78 wherein the second oligonucleotide component is directly labelled with the second detection moiety.

[0208] 80. The method of any one of embodiments 57 to 79 wherein the first fragment is not directly labelled with a detection moiety.

[0209] 81. The method of any one of embodiments 57 to 79 wherein the second fragment is not directly labelled with the first detection moiety; or wherein the first oligonucleotide component is not directly labelled with the first detection moiety.

[0210] 82. The method of any one of embodiments 57 to 81 wherein the second capture region of the first oligonucleotide component is capable of hybridisation to a third capture region of the first oligonucleotide component by complementary base pairing to form the second double-stranded portion.

[0211] 83. The method of any one of embodiments 57 to 81 wherein the first oligonucleotide further comprises a third capture region capable of hybridisation to the second capture region of the first oligonucleotide component by complementary base pairing to form the second double-stranded portion.

[0212] 84. The method of any one of embodiments 57 to 81 wherein the second capture region of the first oligonucleotide component is capable of hybridisation to a second capture region of the second oligonucleotide component by complementary base pairing to form the second double-stranded portion.

[0213] 85. The method of embodiment any one of embodiments 82 to 84 wherein the first doublestranded portion of the TLM probe has a Tm that is above the first temperature. 86. The method of any one of embodiments 82 to 84 wherein the first double-stranded portion and / or the second double-stranded portion of the TLM probe have a Tm that is below the second temperature.

[0214] 87. The method of any one of embodiments 82 to 86 wherein the Tm of the first doublestranded portion is higher than the Tm of the second double-stranded portion.

[0215] 88. The method of any one of embodiments 82 to 86 wherein the Tm of the second doublestranded portion is less than the Tm of the first double-stranded portion.

[0216] 89. The method of any one of embodiments 57 to 80 wherein the first oligonucleotide component is directly labelled with the first detection moiety.

[0217] 90. The method of any one of claims 57 to 80 wherein the TLM probe does not comprise more than two detection moieties.

[0218] 91. The method of any one of embodiments 57 to 90 wherein the first oligonucleotide component is not directly labelled with more than one detection moiety.

[0219] 92. The method of any one of embodiments 57 to 91 wherein the first oligonucleotide component is not connected to more than one detection moiety.

[0220] 93. The method of any one of embodiments 57 to 92 wherein the second oligonucleotide component is not directly labelled with more than one detection moiety.

[0221] 94. The method of any one of embodiments 57 to 93 wherein the second oligonucleotide component is not connected to more than one detection moiety.

[0222] 95. The method of any one of embodiments 57 to 94 wherein the second nucleic acid probe is directly labelled with the third and fourth detection moieties.

[0223] 96. The method of any one of embodiments 57 to 94 wherein the second nucleic acid probe is not directly labelled with the third and fourth detection moieties.

[0224] 97. The method of any one of embodiments 57 to 96 wherein:

[0225] the first detection moiety is a fluorophore, and the second detection moiety is a quencher; or

[0226] the first detection moiety is a quencher, and the second detection moiety is a fluorophore.

[0227] 98. The method of any one of embodiments 57 to 97 wherein:

[0228] the first detection moiety is a fluorophore, and the second detection moiety is a quencher; or

[0229] the first detection moiety is a quencher, and the second detection moiety is a fluorophore;

[0230] and wherein the TLM probe does not comprise more than one quencher.

[0231] 99. The method of any one of embodiment 57 to 98 wherein: the first detection moiety is a fluorophore, and the second detection moiety is a quencher; or

[0232] the first detection moiety is a quencher, and the second detection moiety is a fluorophore;

[0233] and wherein the first detectable signal is fluorescence emitted in the presence of the first target.

[0234] 100. The method of any one of embodiments 57 to 99 wherein neither the first oligonucleotide component, the second oligonucleotide component nor the third oligonucleotide component serve as a primer for a DNA polymerase in an extension reaction.

[0235] 101. The method of any one of embodiments 57 to 100 wherein neither the first oligonucleotide component, the second oligonucleotide component nor the third oligonucleotide component serve as a template for a DNA polymerase in an extension reaction.

[0236] 102. The method of any one of embodiments 57 to 101 wherein neither the second oligonucleotide component nor the third oligonucleotide component is enzymatically cleaved or degraded.

[0237] 103. The method of any one of embodiments 57 to 102 wherein the sensor region is located between the first capture region of the first oligonucleotide and the first detection moiety or between the second capture region of the first oligonucleotide component and the third capture region of the first oligonucleotide component.

[0238] 104. The method of any one of embodiments 57 to 103 wherein following said treating the first fragment is capable of hybridising to the second oligonucleotide component via the first capture region.

[0239] 105. The method of any one of embodiments 57 to 103 wherein the second capture region of the first oligonucleotide is capable of hybridisation to a third capture region of the first oligonucleotide component by complementary base pairing to form a second doublestranded portion, and the probe further comprises a third oligonucleotide component comprising a first capture region capable of hybridisation to a fourth capture region of the first oligonucleotide component by complementary base pairing to form a third doublestranded portion.

[0240] 106. The method of any one of embodiments 57 to 103 wherein the second capture region of the first oligonucleotide is capable of hybridisation to a second capture region of the second oligonucleotide component by complementary base pairing to form a second double-stranded portion, and the probe further comprises a third oligonucleotide component comprising a first capture region capable of hybridisation to a third capture region of the first oligonucleotide component by complementary base pairing to form a third double-stranded portion.

[0241] 107. The method of embodiment 105 or 106 wherein the third oligonucleotide is connected to a detection moiety or wherein the third oligonucleotide is directly labelled with a detection moiety.

[0242] 108. The method of embodiment 107 wherein the detection moiety is the first or the second detection moiety, optionally wherein the first detection moiety is a fluorophore, and the second detection moiety is a quencher; or the first detection moiety is a quencher, and the second detection moiety is a fluorophore.

[0243] 109. The method of any one of embodiments 57 to 108 wherein:

[0244] the third detection moiety is a fluorophore, and the fourth detection moiety is a quencher; or

[0245] the third detection moiety is a quencher, and the fourth detection moiety is a fluorophore.

[0246] 110. The method of any one of embodiments 57 to 109 wherein the first target is a nucleic acid and at least a portion of the sensor region hybridises to a complementary sequence in the first target to thereby form a duplex between the sensor region and the first target. 111. The method of embodiment 110 wherein the first enzyme is an endonuclease that recognises a sequence in the duplex, preferably wherein the endonuclease digests the duplex to thereby form the first and second fragments, more preferably wherein the endonuclease is a nicking endonuclease that digests the sensor region of the first oligonucleotide component after formation of the duplex to thereby form the first and second fragments.

[0247] 112. The method of embodiment 110 wherein the first enzyme is an exonuclease that hydrolyses the sensor region of the first oligonucleotide component after formation of the duplex to thereby form the first and second fragments, optionally wherein the exonuclease is a polymerase with exonuclease activity.

[0248] 113. The method of embodiment 112 wherein

[0249] - the first target is a nucleic acid,

[0250] - at least a portion of the sensor region hybridises to a complementary sequence in the first target to thereby form a duplex between the sensor region and the first target,

[0251] - said mixture further comprises a first target primer capable of binding to the first target at a region upstream of said complementary sequence,

[0252] - said treating the mixture comprises: hybridisation of the first target primer to the first target by complementary base pairing, extending the primer using the polymerase with exonuclease activity and using the first target as a template,

[0253] wherein the polymerase comprising exonuclease activity digests the sensor region of the first oligonucleotide component after formation of the duplex.

[0254] 114. The method of any one of embodiments 57 to 110 wherein the first enzyme is a DNAzyme.

[0255] 115. The method of any one of embodiments 57 to 110 wherein the first target is a nucleic acid and the sensor region of the first oligonucleotide component is not complementary to the first target or wherein the first target is a nucleic acid and the first enzyme is a first target multi-component nucleic acid enzyme (MNAzyme) comprising two partzyme oligonucleotides capable of self-assembling to form the first target MNAzyme only in the presence of the first target.

[0256] 116. The method of embodiment 115 wherein said treating comprises:

[0257] hybridising sensor arms of the first target MNAzyme to the first target by complementary base pairing, and

[0258] hybridising substrate arms of the first target MNAzyme to at least a portion of the sensor region of the first oligonucleotide component by complementary base pairing to facilitate cleavage of the first oligonucleotide component and generation of the first and second fragments.

[0259] 117. The method of any one of embodiments 57 to 116 wherein:

[0260] - the second nucleic acid probe is a substrate for a second target multi-component nucleic acid enzyme (MNAzyme), the second target MNAzyme comprising two partzyme oligonucleotides capable of self-assembling to form the second target MNAzyme only in the presence of the second target;

[0261] - the mixture further comprises:

[0262] the second target MNAzyme capable of cleaving the second nucleic acid probe only when the second target is present in the sample;

[0263] - said treating further comprises:

[0264] hybridising sensor arms of the second target MNAzyme to the second target by complementary base pairing, and

[0265] hybridising substrate arms of the second target MNAzyme to the second nucleic acid probe by complementary base pairing to facilitate cleavage of the second nucleic acid probe thereby providing said modification to the second nucleic acid probe and enabling the third and fourth detection moieties to spatially separate and generate the second detectable signal.

[0266] 118. The method of embodiment 117 wherein the second nucleic acid probe is a stem-loop oligonucleotide comprising a double-stranded stem portion of hybridised nucleotides, opposing strands of which are linked by an unbroken single-stranded loop portion of unhybridised nucleotides of which all or a portion of which is complementary to the substrate arms of the second target MNAzyme.

[0267] 119. The method of embodiment 118 wherein the stem-loop oligonucleotide is an intact stem-loop oligonucleotide and said modification comprises cleavage of a sensor region in the loop portion and the formation of a split stem-loop oligonucleotide.

[0268] 120. The method of any one of embodiments 57 to 116 wherein:

[0269] - the second target is a nucleic acid,

[0270] - the second nucleic acid probe is a stem-loop oligonucleotide comprising a double-stranded stem portion of hybridised nucleotides opposing strands of which are linked by an unbroken single-stranded loop portion of unhybridised nucleotides of which all or a portion is complementary to the second target,

[0271] - the mixture further comprises a polymerase with exonuclease activity,

[0272] - said treating the mixture comprises using conditions suitable for:

[0273] hybridisation of the second target to the single-stranded loop portion of the stem-loop oligonucleotide by complementary base pairing to form a first double-stranded sequence comprising a portion of the second target,

[0274] hybridisation of a primer to the second target to form a second double-stranded sequence located upstream relative to the first double-stranded sequence comprising the portion of the second target,

[0275] extending the primer using the polymerase with exonuclease activity and using the second target as a template,

[0276] wherein the polymerase comprising exonuclease activity digests the single-stranded loop portion of the first double-stranded sequence and thereby forms a split stem-loop oligonucleotide.

[0277] 121. The method of any one of embodiments 57 to 116, wherein:

[0278] - the second target is a nucleic acid,

[0279] - the second nucleic acid probe is a stem-loop oligonucleotide comprising a double-stranded stem portion of hybridised nucleotides opposing strands of which are linked by an unbroken single-stranded loop portion of unhybridised nucleotides of which all or a portion is complementary to the second target, - the mixture further comprises an endonuclease, and

[0280] - said treating the mixture comprises using conditions suitable for:

[0281] hybridisation of the second target to the single-stranded loop portion of the stem-loop oligonucleotide by complementary base pairing to form a double-stranded sequence comprising a portion of the second target,

[0282] association of the endonuclease with the double-stranded sequence comprising a portion of the second target, and

[0283] catalytic activity of endonuclease allowing it to digest the single-stranded loop portion of the double-stranded sequence and thereby form a split stem-loop oligonucleotide.

[0284] 122. The method of any one of embodiments 118 to 121 wherein:

[0285] - the stem portion of the intact stem-loop oligonucleotide has a melting temperature (Tm) that is above the Tm of the stem portion of the split stem-loop oligonucleotide;

[0286] - the first temperature is below the Tm of the stem portion of the intact stem-loop oligonucleotide, and the stem portion of the split stem-loop oligonucleotide;

[0287] - the second temperature is below the Tm of the stem portion of the intact stem-loop oligonucleotide, and is above the Tm of the stem portion of the split stem-loop oligonucleotide; and

[0288] - the first temperature is below the second temperature.

[0289] 123. The method of any one of embodiments 118 to 122 wherein the Tm of the stem portion of the split stem-loop oligonucleotide is above the first temperature.

[0290] 124. The method of any one of embodiments 118 to 123 wherein the Tm of the stem portion of the intact and split stem-loop oligonucleotide(s) is above the Tm of the first doublestranded portion of the TLM probe.

[0291] 125. The method of any one of embodiments 57 to 116 wherein:

[0292] the second nucleic acid probe is a stem-loop oligonucleotide comprising a double-stranded stem portion of hybridised nucleotides opposing strands of which are linked by an unbroken single-stranded loop portion of unhybridised nucleotides of which all or a portion is complementary to the second target, and wherein

[0293] the modification of the second nucleic acid probe is a conformational change arising from hybridisation of the second target to the single-stranded loop portion by complementary base pairing that causes spatial separation of the third and fourth detection moieties.

[0294] 126. The method of embodiment 125 wherein the conformational change is dissociation of the opposing strands in the double-stranded stem portion of the second nucleic acid probe. 127. The method of any one of embodiments 118 to 126 wherein the third and fourth detection moieties are connected to opposing strands of the double-stranded stem portion of the second nucleic acid probe.

[0295] 128. The method of any one of embodiments 57 to 116, wherein:

[0296] - the second target is a nucleic acid,

[0297] - the mixture further comprises:

[0298] a primer complementary to a first sequence in the second target, a pitcher oligonucleotide comprising a region complementary to a second sequence in the second target that differs from the first sequence, and a tag portion that is not complementary to the second target,

[0299] a first polymerase comprising exonuclease activity, and

[0300] optionally a second polymerase, and

[0301] - said treating the mixture comprises:

[0302] conditions suitable to hybridise the primer and the pitcher oligonucleotide to the second target,

[0303] extending the primer using the first or second polymerase and the second target as a template to thereby cleave off the tag portion,

[0304] hybridising the cleaved tag portion to the second nucleic acid probe by complementary base pairing,

[0305] and extending the tag portion using the polymerase and the second nucleic acid probe as a template to generate a double-stranded catcher sequence comprising the second nucleic acid probe thereby providing said modification to the second nucleic acid probe and enabling the third and fourth detection moieties to provide the second detectable signal.

[0306] 129. The method of embodiment 128 wherein:

[0307] - the double-stranded catcher sequence has a Tm that is above the first temperature; and - the second temperature is below the Tm of the double-stranded catcher sequence.

[0308] 130. The method of embodiment 128 or embodiment 129 wherein said extending the tag portion spatially separates the third and fourth detection moieties to thereby generate the second detectable signal.

[0309] 131. The method of any one of embodiments 57 to 116 wherein the second target is a nucleic acid and the second nucleic acid probe is a two-part probe comprising a first part oligonucleotide and a second part oligonucleotide, wherein:

[0310] - the first part oligonucleotide is complementary to a first portion of the second target, - the second part oligonucleotide is complementary to a second portion of the second target, - the first and second portions of the second nucleic acid target flank one another but do not overlap,

[0311] - said treating the mixture comprises:

[0312] forming a duplex structure comprising:

[0313] a first double-stranded portion by hybridising the first part oligonucleotide to the second target by complementary base pairing, and

[0314] a second double-stranded portion by hybridising the second part oligonucleotide to the second target by complementary base pairing,

[0315] thereby bringing the first and second part oligonucleotides into proximity and providing said modification to the second nucleic acid probe enabling the third and fourth detection moieties to come into close proximity and generate the second detectable signal.

[0316] 132. The method of embodiment 131 wherein the second detectable signal is a decrease in fluorescence.

[0317] 133. The method of embodiment 131 wherein the second detectable signal is an increase in fluorescence.

[0318] 134. The method of any one of embodiments 57 to 116, wherein:

[0319] - the second target is a nucleic acid,

[0320] - the second nucleic acid probe comprises a sequence that is complementary to the second target,

[0321] - the mixture further comprises:

[0322] a primer complementary to a portion of the second target, and

[0323] a polymerase with exonuclease activity;

[0324] - said treating the mixture comprises:

[0325] hybridising the primer to the second target by complementary base pairing, hybridising the second nucleic acid probe to the second target by complementary base pairing,

[0326] extending the primer using the polymerase and the second target as a template to thereby digest the second nucleic acid probe and provide said modification to the second nucleic acid probe enabling the third and fourth detection moieties to spatially separate and generate the second detectable signal.

[0327] 135. The method of any one of embodiments 57 to 116, wherein:

[0328] - the second target is a nucleic acid,

[0329] - the mixture further comprises a restriction endonuclease capable of digesting a doublestranded duplex comprising the second target; and

[0330] - said treating the mixture comprises: hybridising the second nucleic acid probe to the second target by complementary base pairing to thereby form the double-stranded duplex,

[0331] digesting the duplex using the restriction endonuclease to thereby provide said modification to the second nucleic acid probe and enabling the third and fourth detection moieties to spatially separate and generate the second detectable signal.

[0332] 136. The method of any one of embodiments 57 to 116 wherein:

[0333] - the second nucleic acid probe is a second TLM probe comprising a first oligonucleotide component and a second oligonucleotide component,

[0334] wherein the first oligonucleotide component of the second TLM probe comprises a first capture region and a second capture region, the first capture region being capable of hybridisation to a first capture region of the second oligonucleotide component of the second TLM probe by complementary base pairing to form a first double-stranded portion, and the second capture region being capable of hybridisation to a third capture region of the first oligonucleotide component of the second TLM probe or to a second capture region of the second oligonucleotide component of the second TLM probe by complementary base pairing to form a second double-stranded portion,

[0335] wherein the first oligonucleotide component of the second TLM probe further comprises a single-stranded loop portion of unhybridised nucleotides comprising a sensor region capable of serving as a substrate for an enzyme only when the second target is present in the sample, wherein the sensor region is located between the first and second capture regions of the first oligonucleotide component

[0336] wherein the first oligonucleotide component of the second TLM probe is connected to the third detection moiety and the second oligonucleotide component of the second TLM probe is connected to the fourth detection moiety,

[0337] wherein the third detection moiety of the first oligonucleotide component of the second TLM probe is connected to the second capture region of the first oligonucleotide component of the second TLM probe,

[0338] - the mixture further comprises the second enzyme,

[0339] - said treating of the mixture comprises:

[0340] treating the mixture under conditions suitable for the enzyme to digest the sensor region of the second TLM probe to thereby generate a first fragment comprising the first capture region of the first oligonucleotide and a second fragment connected to the third detection moiety, thereby enabling the third and fourth detection moieties to spatially separate and generate a second detectable signal. 137. The method of embodiment 136 wherein the first enzyme is the same as the second enzyme.

[0341] 138. The method of embodiment 136 or embodiment 137 wherein the double-stranded portions of the second TLM probe have a Tm above the Tm of the first double stranded portion and the second double stranded portion of the first TLM probe.

[0342] 139. The method of any one of embodiments 57 to 138 wherein:

[0343] - the first target is a nucleic acid;

[0344] - the second target is a nucleic acid; or

[0345] - the first target is a nucleic acid and the second target is a nucleic acid.

[0346] 140. The method of embodiment 139 wherein the first target and / or the second target is an amplicon of a nucleic acid.

[0347] 141. The method of embodiment 140 wherein the amplicon is produced by an amplification reaction selected from the group consisting of polymerase chain reaction (PCR), strand displacement amplification (SDA), nicking enzyme amplification reaction (NEAR), helicase dependent amplification (HD A), Recombinase Polymerase Amplification (RPA), loop-mediated isothermal amplification (LAMP), rolling circle amplification (RCA), transcription-mediated amplification (TMA), self-sustained sequence replication (3 SR), nucleic acid sequence based amplification (NASBA), Ligase Chain Reaction (LCR) or Ramification Amplification Method (RAM) and reverse transcription polymerase chain reaction (RT-PCR).

[0348] 142. The method of embodiment 141 wherein said determining:

[0349] - occurs prior to said amplification or within 1, 2, 3, 4, or 5 cycles of said amplification commencing; and / or

[0350] - occurs after completion of said amplification.

[0351] 143. The method of any one of embodiments 140 to 142 wherein said determining the presence or absence of the first and second targets comprises a melt curve analysis.

[0352] 144. The method of any one of embodiments 57 to 116 or 136 to 143 wherein:

[0353] - the mixture further comprises a DNAzyme or a ribozyme requiring a co-factor for catalytic activity;

[0354] - said treating of the mixture comprises using conditions suitable for:

[0355] binding of the co-factor to the DNAzyme or ribozyme to render it catalytically active, hybridisation of the DNAzyme or ribozyme to the second nucleic acid probe by complementary base pairing, and

[0356] catalytic activity of the DNAzyme or ribozyme to thereby digest the second nucleic acid probe and thereby provide said modification to the second nucleic acid probe enabling the third and fourth detection moieties to spatially separate and generate the second detectable signal, and

[0357] - the second target is the co-factor.

[0358] 145. The method of any one of embodiments 57 to 110 or 117 to 144 wherein:

[0359] - the first enzyme is a DNAzyme or a ribozyme requiring a co-factor for catalytic activity,

[0360] - said treating of the mixture comprises using conditions suitable for:

[0361] binding of the cofactor to the DNAzyme or ribozyme to render it catalytically active, hybridisation of the DNAzyme or ribozyme to the first oligonucleotide component by complementary base pairing,

[0362] catalytic activity of the DNAzyme or ribozyme to thereby digest the first oligonucleotide component and generate the first fragment and the second fragment, and

[0363] - the first target is the co-factor.

[0364] 146. The method of embodiment 144 or embodiment 145 wherein the co-factor is a metal ion, such as a metal ion selected from: Mg2+, Mn2+, Ca2+ and Pb2+.

[0365] 147. The method of any one of embodiments 57 to 106 or 117 to 144 wherein the first enzyme is an aptazyme wherein:

[0366] - the sensor region comprises a substrate for an aptazyme;

[0367] - the first target is an analyte, protein, peptide, compound or nucleic acid;

[0368] - the mixture comprises an aptazyme comprising an aptamer capable of binding to the first target; and

[0369] - said treating the mixture further comprises binding of the aptazyme to the first target and to the sensor region to facilitate cleavage of the first oligonucleotide component to thereby generate the first fragment and the second fragment.

[0370] 148. The method of any one of embodiments 57 to 147 wherein generation of the first detectable signal is not reversible at the first temperature.

[0371] 149. The method of any one of embodiments 57 to 148 wherein the first temperature is lower than the second temperature or wherein the first temperature is higher than the second temperature.

[0372] 150. The method of any one of embodiments 57 to 149 wherein the first enzyme does not digest the first target and / or the second target.

[0373] 151. The method of any one of embodiments 57 to 150 wherein the first temperature differs from the second temperature by more than: 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C or 60°C. 152. The method of any one of embodiments 57 to 150 wherein the first temperature differs from the second temperature by at least 16°C.

[0374] 153. The method of any one of embodiments 57 to 152 wherein the biological sample is obtained from a subject and / or wherein the method is performed in vitro or ex vivo.

[0375] 154. The method of any one of embodiments 57 to 153 wherein the presence or absence of the first target in a sample is determined at temperatures above the melting temperature (Tm) of the cleaved TLM probe and below the Tm of the intact TLM probe.

[0376] 155. A composition comprising:

[0377] a temperature-dependent looped multi-component (TLM) probe comprising a first oligonucleotide component and a second oligonucleotide component,

[0378] wherein the first oligonucleotide component comprises a first capture region and a second capture region, the first capture region being capable of hybridisation to a first capture region of the second oligonucleotide component by complementary base pairing to form a first double-stranded portion, and the second capture region being capable of hybridisation to a third capture region of the first oligonucleotide component or to a second capture region of the second oligonucleotide component by complementary base pairing to form a second double-stranded portion;

[0379] wherein the first oligonucleotide component is connected to a first detection moiety and the second oligonucleotide component is connected to a second detection moiety;

[0380] wherein:

[0381] wherein the first oligonucleotide component further comprises a single-stranded loop portion of unhybridised nucleotides comprising a sensor region capable of serving as a substrate for an enzyme, wherein the sensor region is located between the first and second capture regions of the first oligonucleotide component;

[0382] wherein digestion of the sensor region by the enzyme generates a first fragment and a second fragment;

[0383] wherein the first detection moiety of the first oligonucleotide is connected to the second capture region of the first oligonucleotide.

[0384] 156. The composition of embodiment 155 wherein:

[0385] the TLM probe does not comprise more than two detection moieties;

[0386] the first oligonucleotide component is not connected to more than one detection moiety; or the first fragment is not directly labelled with a detection moiety.

[0387] 157. The composition of embodiment 155 or 156, further comprising an enzyme capable of modifying the sensor region of the first oligonucleotide component only when the target is present in the sample. 158. The composition of any one of embodiments 155 to 157 wherein the second oligonucleotide component is not directly labelled with more than one detection moiety or wherein the second oligonucleotide component is not connected to more than one detection moiety.

[0388] 159. The composition of any one of embodiments 155 to 157 wherein the second oligonucleotide component is directly labelled with the second detection moiety or wherein the second fragment is not directly labelled with the first detection moiety.

[0389] 160. The composition of any one of embodiments 155 to 159 wherein the first oligonucleotide component is not directly labelled with the first detection moiety.

[0390] 161. The composition of any one of embodiments 155 to 160 wherein the second capture region of the first oligonucleotide is capable of hybridisation to a third capture region of the first oligonucleotide component by complementary base pairing to form a second double-stranded portion, and the probe further comprises a third oligonucleotide component comprising a first capture region capable of hybridisation to a fourth capture region of the first oligonucleotide component by complementary base pairing to form a third double-stranded portion.

[0391] 162. The composition of any one of embodiments 155 to 160 wherein the second capture region of the first oligonucleotide is capable of hybridisation to a second capture region of the second oligonucleotide component by complementary base pairing to form a second double-stranded portion, and the probe further comprises a third oligonucleotide component comprising a first capture region capable of hybridisation to a third capture region of the first oligonucleotide component by complementary base pairing to form a third double-stranded portion.

[0392] 163. The composition of embodiment 161 or 162 wherein the first oligonucleotide component is connected to the first detection moiety via the third oligonucleotide component.

[0393] 164. The composition of embodiment 163 wherein the first capture region differs in length or sequence from the second capture region.

[0394] 165. The composition of any one of embodiments 155 to 159 wherein the first oligonucleotide component is directly labelled with the first detection moiety.

[0395] 166. The composition of any one of embodiments 155 to 165 wherein:

[0396] the first detection moiety is a fluorophore, and the second detection moiety is a quencher; or

[0397] the first detection moiety is a quencher, and the second detection moiety is a fluorophore. 167. The composition of any one of embodiments 155 to 166 wherein:

[0398] the first detection moiety is a fluorophore, and the second detection moiety is a quencher; or

[0399] the first detection moiety is a quencher, and the second detection moiety is a fluorophore;

[0400] and wherein the TLM probe does not comprise more than one quencher.

[0401] 168. The composition of any one of embodiments 155 to 167 wherein the sensor region is located between the first capture region and the first detection moiety.

[0402] 169. The composition of any one of embodiments 155 to 168 wherein following digestion of the sensor region the first fragment is capable of hybridising to the second oligonucleotide component via the first capture region.

[0403] 170. The composition of any one of embodiments 155 to 169, further comprising a multicomponent nucleic acid enzyme (MNAzyme) comprising two partzyme oligonucleotides, each partzyme oligonucleotide having a substrate arm capable of hybridising to at least a portion of the sensor region of the first oligonucleotide component only in the presence of a target.

[0404] 171. The composition of embodiment 170 wherein the substrate arms of the two partzyme oligonucleotides are hybridised to the sensor region of the first oligonucleotide component.

[0405] 172. The composition of any one of embodiments 155 to 171, further comprising a DNAzyme capable of cleaving the sensor region of the first oligonucleotide component only in the presence of a target.

[0406] 173. The composition of any one of embodiments 155 to 172, further comprising an aptazyme capable of cleaving the sensor region of the first oligonucleotide component only in the presence of a target.

[0407] 174. The composition of any one of embodiments 155 to 173, further comprising a restriction endonuclease capable of cleaving the sensor region of the first oligonucleotide component only in the presence of a nucleic acid target.

[0408] 175. The composition of any one of embodiments 155 to 174, further comprising an exonuclease capable of digesting the sensor region of the first oligonucleotide component only in the presence of a nucleic acid target.

[0409] 176. The composition of embodiment 175, wherein the exonuclease is a polymerase with exonuclease activity.

[0410] Brief Description of the Drawings Preferred embodiments of the present invention will now be described, by way of example only, with reference to the accompanying Figures as set out below.

[0411] Figure 1 Exemplary structures of intact and cleaved TLM probes with one capture region in Oligo 2 and three capture regions in Oligo 1. Oligo 1 also comprises of a sensor region that is capable of being modified by enzymatic activity, (i) An example of an intact TLM probe comprising of one capture region in Oligo 2 and three capture regions in Oligo 1. The hybridised capture regions 2 and 3 in Oligo 1 form an internal hairpin structure. Full separation of fluorophore and quencher labels requires dissociation of the capture region 1 only, hence the effective Tm of an intact probe is Tm of capture region 1. (ii) An example of a cleaved TLM probe comprising of one capture region in Oligo 2 and three capture regions in Oligo 1. Full separation of fluorophore and quencher labels requires dissociation of the hybridised capture region 2 / 3 only, hence the effective Tm of a cleaved probe is Tm of Oligo 1 capture region 2 / 3.

[0412] Figure 2 Exemplary structures of intact and cleaved TLM probes with two capture regions in each Oligo 1 and Oligo 2. Oligo 1 also comprises of a sensor region that is capable of being modified by enzymatic activity, (i) An example of an intact TLM probe comprising of two capture regions between Oligo 1 and Oligo 2. Full separation of fluorophore and quencher labels requires dissociation of both capture regions, hence the effective Tm of an intact probe is the combined Tm of the capture regions, (ii) An example of a cleaved TLM probe comprising two capture regions between Oligo 1 and Oligo 2. Full separation of fluorophore and quencher labels requires dissociation of the capture region 2 only, hence the effective Tm of a cleaved probe is the Tm of capture region 2.

[0413] Figure 3 Exemplary components of TLM probe, PlexZymes and complexes formed in the presence of a target, (i) The components for a TLM may include an Oligo 1 comprising a sensor region that serves as a substrate for a PlexZyme and a quencher at one terminus, and an Oligo 2 labelled with a fluorophore which is capable of hybridising to the Oligo 1. (ii) The PlexZyme may include two components, PartZymes A and B, each of which has one region complementary to the target known as a target binding arm, a second region complementary to a PlexZyme substrate known as a substrate binding arm, and an intervening region which constitutes half of a catalytic core sequence, (iii) A target sequence for detection, (iv) A fully assembled PlexZyme bound to a TLM probe. When the PartZyme A and B target binding arms hybridise adjacently on a target a catalytic enzyme complex known as a PlexZyme forms. PlexZyme substrate sequences within the sensor region of the Oligo 1 can then hybridise to substrate binding arms of the PlexZyme. The PlexZyme is capable of cleaving TLM probes in a multiple turnover manner. Figure 4 Exemplary TLM Probes suitable for cleavage by a PlexZyme (i.e., MNAzyme) assembled in the presence of a target. The TLM probe has two oligonucleotide components namely: a first oligonucleotide (Oligo 1) comprising a sensor region serving as a PlexZyme substrate, and a quencher at one terminus; and a second oligonucleotide (Oligo 2) labelled with a fluorophore. The TLM probe structure may present in different forms ((i) or (iii)) as depicted in Figure 1 and 2 respectively. The fluorophore can be positioned on Oligo 2 and the quencher on Oligo 1 as in (i) and (iii), or the fluorophore can be positioned on Oligo 1 and the quencher on Oligo 2 as in (ii) and (iv).

[0414] Figure 5 Exemplary structures formed by components of a TLM probe and fluorescence at various temperatures in the presence and absence of target. In all panels, uncleaved or cleaved Oligo 1 is black and labelled with a quencher (Q); Oligo 2 is grey and labelled with a fluorophore (F) which is either in a quenched state (circles) or an unquenched / fluorescent state (stars). Partzymes, unassembled or assembled into PlexZymes, are grey; target nucleic acids are white with a grey outline; Low temperatures are below effective Tmcleaved, middle temperatures are above effective Tmcleavedand below effective Tmintact whilst high temperatures are above the effective Tmintact. During the course of a reaction, at the middle temperature, there will be an increase in fluorescence in presence of target (ii) but no increase in fluorescence in absence of target (v). At the low and high temperature, the level of fluorescence is equal regardless of the presence (i, iii) or absence (iv, vi) of target. The fluorescence measured provides a background reading which does not change during the reaction. Panel (i) illustrates reaction components at the low temperature in the presence of target. PartZymes assemble into PlexZymes which cleave the Oligo 1 generating a first fragment and a second fragment. At this temperature, the two Oligo 1 fragments remain hybridised through Oligo 1 capture region 2 and 3. Panel (iv) illustrates reaction components at the low temperature in the absence of target. PartZymes are free in solution and uncleaved Oligo 1 remains quenched due to close proximity of the F and Q since the probes are fully hybridised. Panel (ii) illustrates reaction components at the middle temperature in the presence of target. PartZymes in general do not assemble into PlexZymes once the temperature has increased over a certain point; however, when cleavage has previously occurred at the low temperature both cleaved and uncleaved Oligo Is will be present in the mix. At the temperatures above effective Tmcleavedand below effective Tmintact, a fluorescence signal is generated due to separation of fluorophore and quencher. Panel (v) illustrates reaction components at the middle temperature in the absence of target. Since intramolecular bonds are stronger than intermolecular bonds, the Tm of the hairpin structure is higher than the effective Tmcleaved, and the probe remains fully assembled at this temperature Panel (iii) illustrates reaction components at the high temperature in the presence of target. All probe components are free in solution, including cleaved and uncleaved Oligo Is. The background fluorescent signal is present due to separation of the F and Q. Panel (iv) illustrates reaction components at the high temperature in the absence of target. PartZymes are free in solution and uncleaved Oligo Is will not be hybridised to the Oligo 2. A background fluorescent signal is present due to separation of the F and Q.

[0415] Figure 6 Panels (i), (ii), (iii), (iv), (v) and (vi) as previously described in Figure 5.

[0416] The middle panels show exemplary amplification plot for PCR where fluorescence (y-axis) is plotted against PCR cycle number (x-axis). The center middle panel shows fluorescent signal obtained at the middle temperature (above effective Tmcleavedand below effective Tmintact) in reactions containing high (A) or low (B) number of copies of target, or when no target (C) is present. The top and bottom middle panel shows fluorescent signal obtained at the low and high temperature respectively (below the effective Tmcleavedor above the effective Tmintact) in the same reactions.

[0417] Figure 7 illustrates four types of general designs for PlexZyme substrate probes which can be used alone or in conjunction with each other to facilitate the detection of target nucleic acids. All four probe types can be cleaved by PlexZymes in the presence of target resulting in an increase in fluorescence. Panel (i) illustrates a “standard” linear PlexZyme substrate which is dual labelled with a fluorophore (F) at one end and a quencher (Q) at the other. When linear probes are cleaved an increase in fluorescence above background can be observed across a broad temperature range (at any given temperature). Panel (ii) illustrates a hairpin stem-loop PlexZyme substrate (LOCS probe) which incorporates a PlexZyme substrate region in the loop, and a stem region labelled with a fluorophore (F) at one end and a quencher (Q) at the other. When LOCS probes are cleaved, an increase in fluorescence above background can only be observed at ‘high’ temperatures which are above the Tm of the stem region of a cleaved, split LOCS probe but below the Tm of the stem region of an intact LOCS probe. Panel (iii) illustrates an M-Tec-P probe which consists of two oligonucleotide components. First oligonucleotide component (OC1) incorporates an internal stem-loop structure, a PlexZyme substrate region in the loop, a capture region and a label. Second oligonucleotide component (OC2) incorporates a region capable of hybridising to the capture region of OC1, and a label. When M-Tec-P probes are cleaved, an increase in fluorescence is only observed at Tow’ temperatures which are below Tm of OC1 / OC2. Panel (iv) illustrates an exemplary TLM probe as described in Figures 1-6. These probes only result in an increase in fluorescence above background at ‘middle’ temperatures above effective Tmcleavedand below effective Tmintact. Figure 8A and 8B schematically illustrate an approach for multiplex analysis of three targets using the combination of one M-Tec-P Probe (A), TLM Probe (B) and one LOCS probe (C) all of which are labelled with the same fluorophore (F) and quencher (Q). Reaction mixes contain an intact M-Tec-P (Ai), an intact TLM Probe (Bi) and an intact LOCS probe (Ci). In the presence of target 1 (Tl), PlexZyme 1 (Pl) assembles and cleaves Ai to generate a cleaved M-Tec-P Probe (Ac) (Figure 8A panel (i)). In the presence of target 2 (T2), PlexZyme 2 (P2) assembles and cleaves Bi to generate a cleaved, split TLM Probe (Be) (Figure 8A panel (ii)). In the presence of target 3 (T3), PlexZyme 3 (P3) assembles and cleaves Ci to generate a cleaved, split LOCS Probe (Cc) (Figure 8A panel (iiii)). Figure 8B panel (i) illustrates structures which can form at temperature 1 which is below the Tm OC1 / OC2 of both Ai and Ac, below effective Tmcleavedof the Be and below the Tm of the stem of both the Cc and Ci. Figure 8B panel (ii) illustrates structures which can form at temperature 2 which is above the Tm OC1 / OC2 of both Ai and Ac, above effective Tmcleavedof Be and below effective Tmintact of Bi and below the Tm of the stem of both the Cc and Ci. Figure 8B panel (iii) illustrates structures which can form at temperature 3 which is above the Tm OC1 / OC2 of both Ai and Ac, above the effective Tmintact of Bi and above the Tm of the stem of the Be but below the Tm of the stem of the Bi. At temperature 1, fluorescence above background is generated in the presence of target 1 (Tl) but not in the absence of TL At temperature 1, Bi and Ci may get cleaved but no fluorescence above background is generated from Be or Cc since the probes remains hybridised and the fluorophore remains quenched. Hence an increase in fluorescence above background at temperature 1 indicates the presence of Tl and background fluorescence is the same regardless of the presence or absence of T2 and T3. At temperature 2, the OC1 and OC2 of both Ai and Ac complexes dissociate and generate background fluorescence only. Bi remains hybridised, while the Oligo 1 fragments of Be dissociate, resulting in an increase in fluorescence above background. At temperature 2, Ci may get cleaved but no fluorescence above background is generated from Cc since the stem remains hybridised and the fluorophore remains quenched. Hence an increase in fluorescence above background at temperature 2 indicates the presence of T2 and background fluorescence is the same regardless of the presence or absence of Tl and T3. At temperature 3, the OC1 and OC2 of both Ai and Ac complexes dissociate and generate background fluorescence only. Similarly, both Bi and Be dissociate and generate background fluorescence only. At temperature 3, the stem of Cc, but not Ci, dissociates resulting in an increase in fluorescence above background. Hence an increase in fluorescence above background at temperature 3 indicates the presence of T3 and background fluorescence is the same regardless of the presence or absence of T1 and T2.

[0418] Figure 9 Illustration of two exemplary TLM probes that could be used for a single channel multiplexing. Both TLM probe A and TLM probe B could be composed of two oligonucleotides (Oligo 1 and Oligo 2) with both probes labelled with the same fluorophore (F) and quencher (Q) dye pair. Each of the Oligo 1 within TLM probe A and TLM probe B could contain difference substrates specific for two PlexZymes capable of assembling in the presence of a first target (Tl) or a second target (T2) respectively. The Tmcleavedand Tmintact of the TLM probe A would be lower than those of TLM probe B.

[0419] Figure 10 An exemplary stem-loop LOCS reporter and its melting temperatures (Tm) in the Intact and Split conformations are illustrated. A LOCS reporter as exemplified can be used in combination with TLM probes, M-Tec probes and various standard reporter probes and substrates well known in the art for detection of nucleic acids. Exemplary intact LOCS reporters (Figure 10A, LHS; top and bottom) have a Loop region which can be cleaved or degraded, a Stem region and detection moiety, for example a fluorophore (F) quencher (Q) dye pair. Cleavage or degradation of the Loop region in the presence of target can produce Split LOCS reporter structures (Figure 10B RHS; top and bottom). The melting temperatures of the stem regions of the Intact LOCS (Tm A) is higher than the Tm of the stem regions in Split LOCS (Tm B). As such, the Stem of the Intact LOCS will melt and separate at temperatures at or above Tm A. In contrast, the stem holding the two fragments of the Split LOCS will melt and separate at temperatures at or above Tm B resulting in increased fluorescence.

[0420] Figure 11 illustrates an exemplary strategy for detection of a target using LOCS oligonucleotides which are universal and can be used to detect any target. In this scheme the LOCS oligonucleotide contains a stem region, a fluorophore quencher dye pair and a loop region. The loop region comprises a universal substrate for a catalytic nucleic acid for example a PlexZyme, also known in the art and referred to herein as a MNAzyme. PlexZymes form when target sensor arms of component partzymes align adjacently on a target. The loop region of the LOCS oligonucleotide binds to the substrate binding arms of the assembled PlexZyme and the substrate within the LOCS loop is cleaved by the PlexZyme to generate a cleaved Split LOCS structure. Both the Intact LOCS and the Split LOCS will be either quenched, or will generate fluorescence, depending upon whether the temperature of the reaction milieu is above or below the melting temperature of their stems, namely Tm A and Tm B respectively. The presence of fluorescence at temperatures between Tm B and Tm A is indicative of the presence of the target which facilitates the cleavage. The target can be directly detected, or target amplicons produced by target amplification protocols, can be detected.

[0421] Figure 12 shows amplification curves where fluorescence was acquired at 45°C, 61 °C and 78°C for reactions containing an TLM-P specific to Chlamydia trachomatis (CT) in the presence of various numbers of copies (10000 (A), 40 (B)) of target or no target (C). A fluorescence signal is only acquired at 61°C, while no signal is observed at 45°C or 78°C.

[0422] Figure 13 shows amplification curves for 1) an TLM Probe specific to human transferrin receptor (TFRC) gene target in the presence of various numbers of copies of target (10000 or 40 per reaction) or no target at three acquisition temperatures 45°C (i), 61 °C (ii) and 78°C (iii). 2) a M-Tec Probe specific to human transferrin receptor (TFRC) gene target in the presence of various numbers of copies of target (10000 or 40 per reaction) or no target at three acquisition temperatures 45°C (iv), 61°C (v) and 78°C (vi). 3) a LOCS Probe specific to human transferrin receptor (TFRC) gene target in the presence of various numbers of copies of target (10000 or 40 per reaction) or no target at three acquisition temperatures 45°C (vii), 61 °C (viii) and 78°C (ix). 4) a linear probe specific to human transferrin receptor (TFRC) gene target in the presence of various numbers of copies of target (10000 or 40 per reaction) or no target at three acquisition temperatures 45°C (x), 61 °C (xi) and 78°C (xii). 5) a molecular beacon (MB) probe specific to Trichomonas vaginalis (TV) beta-tubulin (btub) gene target in the presence of various numbers of copies of target (10000 or 40 per reaction) or no target at three acquisition temperatures 45°C (xiii), 61 °C (xiv) and 78°C (xv). 6) a universal molecular beacon (UMB) probe specific to Chlamydia trachomatis (CT) cryptic plasmid (cry) gene target in the presence of various numbers of copies of target (10000 or 40 per reaction) or no target at three acquisition temperatures 45°C (xvi), 61 °C (xvii) and 78°C (xviii).

[0423] Figure 14 shows real time amplification curves generated using a M-Tec probe for the detection of Neisseria gonorrhoeae (NG) and a TLM probe for the detection of Chlamydia trachomatis (CT) with acquisition in the FAM channel at 46°C (A) and 70°C (B) in the presence of reaction mix and 10000 and 40 copies of NG only (black solid line), or CT only (grey solid line), or no template (black dashed line).

[0424] Figure 15 shows real time amplification curves generated using a TLM probe for the detection of Chlamydia trachomatis (CT) and a LOCS probe for the detection of Mycoplasma genitalium (MG) with acquisition in the FAM channel at 58°C (A) and 78°C (B) in the presence of reaction mix and 10000 and 40 copies of CT only (black solid line), or MG only (grey solid line), or no template (black dashed line). Figure 16A, 16B, 17A and 17B show two multiplex reactions where three targets are quantitively detected in a single reaction at the same wavelength during PCR. In Figures 16A and 16B, target 1, Neisseria gonorrhoeas (NG), is detected with an M-Tec- P probe; target 2, Chlamydia trachomatis (CT) is detected with TLM-P probe; and target 3, Mycoplasma genitalium (MG) is detected with a LOCS probe. All three probes are labelled with FAM fluorophore. Amplification plots are shown for reactions mediated by AptaTaq exo DNA polymerase with acquisition at 45°C, 61°C and at 78°C. Reactions contained either 10,000 or 40 copies of target(s) (grey curves) or no target (black curves).

[0425] Figure 16B shows that having all targets present does not affect assay performance; solid grey shows single target and dotted line shows mixed target (targets 1, 2 and 3 at equal concentration).

[0426] In Figures 17A and 17B, target 1, Neisseria gonorrhoeae (NG), is detected with an M-Tec-P probe; target 2, Mycoplasma genitalium (MG) is detected with TLM-P probe; and target 3, Chlamydia trachomatis (CT) is detected with a LOCS probe. All three probes are labelled with AttoRholOl fluorophore. Amplification plots are shown for reactions mediated by AptaTaq exo DNA polymerase with acquisition at 40°C, 61°C and at 78°C. Reactions contained either 10,000 or 40 copies of target(s) (grey curves) or no target (black curves). Figure 17B shows that having all targets present does not affect assay performance; solid grey shows single target and dotted line shows mixed target (targets 1, 2 and 3 at equal concentration).

[0427] Figure 18A and 18B show a multiplex reaction where four targets are quantitively detected in a single reaction at the same wavelength during PCR. Target 1, Neisseria gonorrhoeae (NG), is detected with an M-Tec-P probe; target 2, Mycoplasma genitalium (MG) is detected with first TLM-P probe; target 3, Chlamydia trachomatis (CT) is detected with second TLM-P probe and target 4, Trichomonas vaginalis (TV) is detected with a LOCS probe. Amplification plots are shown for reactions mediated by AptaTaq exo DNA polymerase with acquisition at 34°C, 56°C, 70°C and at 82°C. Reactions contained either 10,000 or 40 copies of target (grey curves) or no target (black curves).

[0428] Figure 18B shows that having all targets present does not affect assay performance; solid grey shows single target and dotted line shows mixed target (targets 1, 2, 3 and 4 at equal concentration).

[0429] Figure 19 shows an end-point qualitative analysis by displaying the difference in fluorescence before and after PCR (ARFU) in reactions containing an M-Tec-P probe for detection of Neisseria gonorrhoeae (NG), an TLM probe for detection of Chlamydia trachomatis (CT) and a LOCS probe for detection of Mycoplasma genitalium (MG) at 45°C (A), 61 °C (B) and 78°C (C). The graphs are displayed as mean of duplicates of reactions containing either 10,000 or 100 copies of NG only, CT only, or MG only, or no target (NTC). The error bars denote the standard deviation.

[0430] Figure 20 shows an end-point qualitative analysis by displaying the difference in fluorescence before and after PCR (ARFU) in reactions containing an M-Tec-P probe for detection of Neisseria gonorrhoeae (NG), first TLM probe for detection of Mycoplasma genitalium (MG), second TLM probe for detection of Chlamydia trachomatis (CT) and a LOCS probe for detection of Trichomonas vaginalis (TV) at 34°C (A), 56°C (B), 70°C (C) and 82°C (D). The graphs are displayed as mean of triplicates of reactions containing either 100,000 or 1000 copies of NG only, MG only, CT only or TV only, or no target (NTC). The error bars denote the standard deviation.

[0431] Figure 21 illustrates analysis of fluorescence data acquired in the FAM channel where TLM-P was used for direct detection of a nucleic acid target (CT), in the absence of target amplification. End-point detection of the target, which was calculated as the difference between the fluorescence signal acquired before and after the isothermal incubation with partzymes specific for the target, showed that there was a significant increase in fluorescence signal only in the presence of the target, but not in the absence at 61 °C (Figure 21B). In contrast, there was no significant increase in fluorescence regardless of the presence or absence of the target at 45°C (Figure 21A) or 78°C (Figure 21C). The derivative melt curve analysis from 40°C to 95°C acquired after the isothermal reaction step (Figure 21D) shows there is a melt signature curve with a peak at ~56°C and ~74°C in reactions with the target template (solid grey line), but there’s no melt signature curve peak at ~56°C observed in the reactions containing the no target template (black solid line).

[0432] Figure 22 illustrates exemplary components and structures for TLM Probe complexes composed of (i) three oligonucleotides, Oligo 1, Oligo 2 and Oligo 3, (ii) Partzymes A and B, and (iii) target template; where (iv) shows the complex formed when an TLM Probe binds to a PlexZyme assembled in the presence of the target. In this complex, Oligo 1 contains a substrate amenable to cleavage by a PlexZyme but it is not directly labelled with a dye. The Oligo 2 and Oligo 3 contain non-target sequences complementary to respective capture regions of Oligo 1 and are labelled with fluorophore and quencher dye pairs. The melting temperature of the complementary capture regions of Oligo 1 and Oligo 2 could be lower than the melting temperature of the complementary capture regions of Oligo 1 and Oligo 3.

[0433] Figure 23 Exemplary structures formed by a three-oligonucleotide TLM and fluorescence at various temperatures in the presence and absence of target. In all panels, uncleaved or cleaved Oligo 1 is black; Oligo 2 is grey and labelled with a fluorophore (F) which is either in a quenched state (circles) or an unquenched / fluorescent state (stars); and Oligo 3 is grey and labelled with a quencher (Q). Partzymes, unassembled or assembled into PlexZymes, are grey; target nucleic acids are white with a grey outline; The low temperature is below effective Tmcleaved, the middle temperature is above effective Tmcleavedand below effective Tmintact whilst the high temperature is above the effective Tmintact. During the course of a reaction, at the middle temperature there will be an increase in fluorescence in presence of target (ii) but no increase in fluorescence in absence of target (v). At the low and high temperature, the level of fluorescence is equal regardless of the presence (i, iii) or absence (iv, vi) of target. The fluorescence measured provides a background reading which does not change during the reaction. Panel (i) illustrates reaction components at the low temperature in the presence of target. At this first temperature, both Oligo 2 and Oligo 3 would bind to the intact Oligo 1 resulting in quenched probe complexes. If Oligo 1 was cleaved by a PlexZyme assembled in the presence of target, Oligo 2 and Oligo 3 would remain bound to fragments of Oligo 1 components; and the Oligo 1 fragments would remain hybridised through the Oligo 1 capture region 2 / 3. At the second (middle) temperature, the two Oligo 1 fragments dissociate, allowing the dye labels to separate and an increase in fluorescence above the baseline would be measurable at this temperature. At a third (high) temperature, Oligo 2 dissociates from both intact Oligo 1 and / or a cleaved Oligo 1 fragments, separating the two dye labels so that a constant level of background would be generated regardless of whether or not the target had been present in the reaction.

[0434] Figure 24 shows an exemplary TLM-H suitable for hydrolysis by exonuclease activity, for example, by inherent 5’ to 3’ exonuclease activity of polymerase in the presence of a target (A) and a TLM-E suitable for hydrolysis by a restriction endonuclease, for example a Nicking enzyme, in the presence of a target (B). Both TLM-H and TLM-E comprise of two oligonucleotides namely a first oligonucleotide (Oligo 1) and a second oligonucleotide (Oligo 2). Oligo 1 is labelled with a quencher at one terminus and contains a sensor region that is complementary to the target. TLM-E further comprises of a double stranded recognition site for the restriction endonuclease. Oligo 2 is labelled with a fluorophore at one terminus and contains a first capture region which is capable of hybridising to the capture region 1 of Oligo 1. Oligo 1 may further comprise of two complementary capture regions forming an internal stem-loop structure, as depicted in this figure, or alternatively a second capture region complementary to a second capture in Oligo 2. Figure 25 Exemplary structures formed by components of an TLM-H and fluorescence at various temperatures in the presence and absence of target. In all panels, unhydrolysed or hydrolysed first oligonucleotide component Oligo 1 is black and labelled with a quencher (Q); second oligonucleotide component Oligo 2 is grey and labelled with a fluorophore (F) which is either in a quenched state (circles) or an unquenched / fluorescent state (stars); PCR primers are grey arrows; target nucleic acids are white with a grey outline. The low temperature is below effective Tmcleaved, the middle temperature is above effective Tmcieaved and below effective Tmintact whilst the high temperature is above the effective Tmintact. During the course of a reaction, at the middle temperature there will be an increase in fluorescence in presence of target (ii) but no increase in fluorescence in absence of target (v). At the low and high temperatures, the level of fluorescence is equal regardless of the presence (i, iii) or absence (iv, vi) of target. The fluorescence measured provides a background reading which does not change during the reaction. Panel (i) illustrates reaction components at the low temperature in the presence of target. In the presence of target, the 5 '-3' exonuclease activity of polymerase hydrolyses the Oligo 1 during PCR resulting in generation of a first fragment and a second fragment. At this temperature, the two Oligo 1 fragments remain hybridised through Oligo 1 capture regions 2 and 3. Panel (iv) illustrates reaction components at the low temperature in the absence of target. No hydrolysis of Oligo 1 occurs and the TLM-H remains quenched due to close proximity of the F and Q. Panel (ii) illustrates reaction components at the middle temperature in the presence of target. In the presence of target, the 5 '-3' exonuclease activity of polymerase may continue to hydrolyse the Oligo 1 during PCR resulting in generation of a first fragment and a second fragment now separate from each other due to the dissociation of Oligo 1 capture regions 2 and 3. This separates the fluorophore from the quencher and gives a fluorescent signal. Panel (v) illustrates reaction components at the middle temperature in the absence of target. No hydrolysis of Oligo 1 occurs and the TLM-H remains quenched due to close proximity of the F and Q as all capture regions remain hybridised. Panel (iii) illustrates reaction components at the high temperature in the presence of target. When a target is present, hydrolysis will have occurred at the lower temperature and both hydrolysed (and residual unhydrolysed) Oligo 1 will be present in the reaction but neither will be hybridised to Oligo 2. The background fluorescent signal is present due to separation of the F and Q. Panel (vi) illustrates reaction components at the high temperature in the absence of target. No hydrolysis of the Oligo 1 occurs, and Oligo 1 and Oligo 2 are not hybridised. A background fluorescent signal is present due to separation of the F and Q. During a reaction, at the middle temperature, there will be an increase in fluorescence in presence of target (ii) but no increase in fluorescence in absence of target (v). At the low and high temperatures, the level of fluorescence is equal regardless of the presence (i,iii) or absence (iv, vi) of target. The fluorescence measured at the low and high temperature provides a background reading which does not change during the reaction.

[0435] Figure 26 schematically illustrates an approach for multiplex analysis of three targets using the combination of one M-Tec-P Probe (A), one TLM-H Probe (B) and one LOCS probe (C) all of which are labelled with the same fluorophore (F) and quencher (Q). Reaction mixes contain an intact M-Tec-H Probe (Ai), an intact TLM-H probe (Bi) and an intact LOCS probe (Ci). In the presence of target 1 (Tl), a PlexZyme (P) assembles and cleaves Ai to generate a cleaved M-Tec-P Probe (Ac). In the presence of target 2 (T2), the 5 '-3 ' exonuclease activity of polymerase hydrolyses Bi to generate a cleaved TLM-H Probe (Be). In the presence of target 3 (T2), a PlexZyme (P) assembles and cleaves Ci to generate a cleaved LOCS Probe (Cc). For temperature-dependent behavior of each probe, refer to Figure 8B. At low temperature, fluorescence above background is generated by Ac in the presence of Tl but not by Ai in the absence of Tl. At this temperature, Bi and Ci may get cleaved but no fluorescence above background is generated from Be or Cc since the hairpin stems remains hybridised and the fluorophore remains quenched. Hence an increase in fluorescence above background at temperature 1 indicates the presence of Tl and background fluorescence is the same regardless of the presence or absence of T2 and T3. At middle temperature, fluorescence above background is generated by Be in the presence of T2 but not by Bi in the absence of T2. At this temperature, Ai and Ci may get cleaved but no fluorescence above background is generated from Ac or Cc; OC1 and OC2 of both Ai and Ac complexes dissociate and generate background fluorescence, and the hairpin stem of Cc remain hybridised and the fluorophore remains quenched. Hence an increase in fluorescence above background at high temperature indicates the presence of T2 and background fluorescence is the same regardless of the presence or absence of Tl and T3. At high temperature, OC1 and OC2 of both Ai / Ac and Oligo 1 and 2 of both Bi / Bc complexes dissociate and generate background fluorescence. At this temperature the stem of Cc, but not Ci, dissociates resulting in an increase in fluorescence above background. Hence an increase in fluorescence above background at high temperature indicates the presence of T3 and background fluorescence is the same regardless of the presence or absence of Tl and T2.

[0436] Figure 27A Exemplary components and structures for TLM-H Probe complexes comprising three oligonucleotides, Oligo 1, Oligo 2 and Oligo 3 together with a 5' primer, a polymerase with inherent 5 '-3' exonuclease activity. In this complex, Oligo 1 contains a sensor region, at least a portion of which is complementary to the target, but it is not directly labelled with a dye. The Oligo 2 and Oligo 3 contain non-target sequences complementary to respective capture regions of Oligo 1 and could be labelled with fluorophore and quencher dye pairs. The melting temperature of the complementary capture regions of Oligo 1 and Oligo 2 could be lower than the melting temperature of the complementary capture regions of Oligo 1 and Oligo 3.

[0437] Figure 27B Exemplary components and structures for TLM-E Probe complexes comprising three oligonucleotides, Oligo 1, Oligo 2 and Oligo 3 together with a nicking endonuclease. In this complex, Oligo 1 contains a sensor region, at least a portion of which is complementary to the target, but it is not directly labelled with a dye. The components Oligo 2 and Oligo 3 would contain non-target sequences complementary to respective capture regions of Oligo 1 and would be labelled with fluorophore and quencher dye pairs. The melting temperature of the complementary capture regions of Oligo 1 and Oligo 2 could be lower than the melting temperature of the complementary capture regions of Oligo 1 and Oligo 3. Hybridisation of the target with the sensor region of Oligo 1 would create the double-stranded recognition sequence for the nicking endonuclease.

[0438] Figure 28 illustrates an example of an TLM-P probe comprising Oligo 1 and Oligo 2, and an Aptazyme. The Oligo 1 oligonucleotides could be designed to contain a quencher, a capture region complementary to a sequence of the Oligo 2 oligonucleotide and a sensor region that may function as a substrate for a specific DNAzyme. The complementary capture region between the Oligo 1 and Oligo 2 oligonucleotides could be designed to have a melting temperature being higher than a first and second temperature, but lower than a third temperature, where the fluorescence measurements would be made at the second temperature. The Oligo 2 oligonucleotide could be designed to contain a fluorophore. The Aptazyme could be designed to contain an aptamer region that could be designed to have a specific affinity to the target, a DNAzyme region that is capable of cleaving the substrate sequence in the sensor region of Oligo 1 oligonucleotide, and a cDNA region containing complementary sequences to the aptamer region. In the absence of target the DNAzyme could be held in an inactive conformation due to binding of the cDNA to the aptamer region. Binding of the specific target to the aptamer could induce dissociation of the cDNA / aptamer double-stranded region, which allows activation of the DNAzyme. This DNAzyme could then cleave the TLM-P probe and generate a detectable signal that is measurable at temperatures above the Tmcleavedand below Tmintact of the TLM-P probe.

[0439] Definitions As used in this application, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the phrase “polynucleotide” also includes a plurality of polynucleotides.

[0440] As used herein, the term “comprising” means “including”. Variations of the word “comprising”, such as “comprise” and “comprises,” have correspondingly varied meanings. Thus, for example, a polynucleotide “comprising” a sequence of nucleotides may consist exclusively of that sequence of nucleotides or may include one or more additional nucleotides.

[0441] As used herein the term “plurality” means more than one. In certain specific aspects or embodiments, a plurality may mean 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or more, and any integer derivable therein, and any range derivable therein.

[0442] As used herein, the term “subject” includes any animal of economic, social or research importance including bovine, equine, ovine, primate, avian and rodent species. Hence, a “subject” may be a mammal such as, for example, a human or a non -human mammal. Also encompassed are microorganism subjects including, but not limited to, bacteria, archaea, viruses, fungi / yeasts, protists and nematodes. A “subject” in accordance with the presence invention also includes infectious agents such as prions. A subject may also include an algae or a plant.

[0443] As used herein, the terms “polynucleotide” and “nucleic acid” may be used interchangeably and refer to a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases, or analogues, derivatives, variants, fragments or combinations thereof, including but not limited to DNA, methylated DNA, alkylated DNA, RNA, methylated RNA, microRNA, siRNA, shRNA, mRNA, tRNA, snoRNA, stRNA, smRNA, pre- and pri-microRNA, other non-coding RNAs, ribosomal RNA, derivatives thereof, amplicons thereof or any combination thereof. By way of non-limiting example, the source of a nucleic acid may be selected from the group comprising synthetic, mammalian, human, animal, plant, fungal, bacterial, viral, archael or any combination thereof.

[0444] As used herein, the term “oligonucleotide” refers to a segment of DNA or a DNA-containing nucleic acid molecule, or RNA or RNA-containing molecule, or a combination thereof. Examples of oligonucleotides include nucleic acid targets; component for M-Tec, substrates, for example, those which can be modified by an PlexZyme; primers such as those used for in vitro target amplification by methods such as PCR; components of PlexZymes; and various other types of reporter probes, including but not limited to, TaqMan or Hydrolysis probes; Molecular Beacons; Binary DNA probes (universal Molecular Beacons); Sloppy Beacons; Eclipse probes; Scorpion Uni-Probe, Scorpion BiProbes primer / probes, Double-stranded probes (Yin-Yang probes), Capture / Pitchers and dual-hybridisation probes. The term “oligonucleotide” includes reference to any specified sequence as well as to the sequence complementary thereto, unless otherwise indicated. Oligonucleotides may comprise at least one addition or substitution, including but not limited to the group comprising 4-acetylcytidine, 5-(carboxyhydroxylmethyl)uridine, 2'-O-methylcytidine, 5-carboxymethylaminomethyl thiouridine, dihydrouridine, 2'-O-methylpseudouridine, beta D-galactosylqueosine, 2'-O-methylguanosine, inosine, N6-isopentenyladenosine, 1 -methyladenosine, 1 -methylpseudouridine, 1 -methylguanosine, 1-methylinosine, 2,2-dimethylguanosine, 2-m ethyladenosine, 2-methylguanosine, 3-methylcytidine, 5-methylcytidine, N6-methyladenosine, 7-m ethylguanosine, 5-methylaminomethyluridine, 5-methoxyaminomethyl-2-thiouridine, beta D-mannosylmethyluridine, 5-methoxycarbonylmethyluridine, 5-methoxyuridine, 2-methylthio-N6-isopentenyladenosine, N-((9-beta-ribofuranosyl-2-methylthiopurine-6-yl)carbamoyl)threonine, N-((9-beta-ribofuranosylpurine-6-yl)N-methyl-carbamoyl)threonine, uridine-5-oxyacetic acid methylester, uridine-5-oxyacetic acid (v), wybutoxosine, pseudouridine, queosine, 2-thiocytidine, 5-methyl-2-thiouridine, 2-thiouridine, 4-thiouridine, 5 -methyluridine, N-((9-beta-D-ribofuranosylpurine-6-yl)carbamoyl)threonine, 2'-O-methyl-5-methyluridine, 2'-O-methyluridine, wybutosine, 3-(3-amino-3-carboxypropyl)uridine, beta D-arabinosyl uridine, beta D-arabinosyl thymidine.

[0445] The terms “polynucleotide” and “nucleic acid” “oligonucleotide” include reference to any specified sequence as well as to the sequence complementary thereto, unless otherwise indicated.

[0446] As used herein, the terms “complementary”, “complementarity”, “match” and “matched” refer to the capacity of nucleotides (e.g. deoxyribonucleotides, ribonucleotides or combinations thereof) to hybridise to each other via Watson-Crick base-pairing, noncanonical base-pairing including wobble base-pairing and Hoogsteen base-pairing (e.g. LNA, PNA or BNA) or unnatural base pairing (UBP). Bonds can be formed via Watson-Crick base-pairing between adenine (A) bases and uracil (U) bases, between adenine (A) bases and thymine (T) bases, between cytosine (C) bases and guanine (G) bases. A wobble base pair is a noncanonical base pairing between two nucleotides in a polynucleotide duplex (e.g. guanine-uracil, inosine-uracil, inosine-adenine, and inosine-cytosine). Hoogsteen base pairs are pairings that, like Watson-Crick base pairs, occur between adenine (A) and thymine (T) bases, and cytosine (C) and guanine (G) bases, but with differing conformation of the purine in relation to the pyrimidine compared to in Watson-Crick base pairings. An unnatural base pair is a manufactured subunit synthesised in the laboratory and not occurring in nature. Nucleotides referred to as “complementary” or that are the “complement” of each other are nucleotides which have the capacity to hybridise together by either Watson-Crick base pairing or by noncanonical base pairing (wobble base pairing, Hoogsteen base pairing) or by unnatural base pairing (UBP) between their respective bases. A sequence of nucleotides that is “complementary” to another sequence of nucleotides herein may mean that a first sequence is 100% identical to the complement of a second sequence over a region of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleotides. Reference to a sequence of nucleotides that is “substantially complementary” to another sequence of nucleotides herein may mean that a first sequence is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the complement of a second sequence over a region of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleotides.

[0447] As used herein, the terms “non-complementary”, “not complementary”, “mismatch” and “mismatched” refer to nucleotides (e.g. deoxyribonucleotides, ribonucleotides, and combinations thereof) that lack the capacity to hybridise together by either Watson-Crick base pairing or by wobble base pairing between their respective bases. A sequence of nucleotides that is “non-complementary” to another sequence of nucleotides herein may mean that a first sequence is 0% identical to the complement of a second sequence over a region of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleotides.

[0448] Reference to a sequence of nucleotides that is “substantially non-complementary” to another sequence of nucleotides herein may mean that a first sequence is less than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% identical to the complement of a second sequence over a region of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleotides.

[0449] As used herein, the term “target” refers to any molecule or analyte present in a sample that the methods of the present invention may be used to detect. The term “target” will be understood to include nucleic acid targets, and non-nucleic acid targets such as, for example proteins, peptides, analytes, ligands, and ions (e.g. metal ions).

[0450] As used herein, an “enzyme” refers to any molecule which can catalyse a chemical reaction (e.g. amplification of a polynucleotide, cleavage of a polynucleotide etc.). Nonlimiting examples of enzymes suitable for use in the present invention include nucleic acid enzymes and protein enzymes. Non-limiting examples of suitable nucleic acid enzymes include ribozymes, MNAzymes (i.e., PlexZymes), DNAzymes and aptazymes. Nonlimiting examples of suitable protein enzymes include exonucleases and endonucleases. The enzymes will generally provide catalytic activity that assists in carrying out one or more of the methods described herein. By way of non-limiting example, the exonuclease activity may be an inherent catalytic activity of, for example, a polymerase. By way of nonlimiting example, the endonuclease activity may be an inherent catalytic activity of, for example, a restriction enzyme including a Nicking endonuclease, a riboendonuclease or a duplex specific nuclease (DSN).

[0451] As used herein, an “amplicon” refers to nucleic acid (e.g. DNA or RNA, or a combination thereof) that is a product of natural or artificial nucleic acid amplification or replication events including, but not limited to PCR, RT-PCR, SDA, NEAR, HD A, RPA, LAMP, RCA, TMA, LCR, RAM, 3 SR, NASBA, and any combination thereof.

[0452] As used herein, the term “stem-loop oligonucleotide” will be understood to mean a DNA or DNA-containing molecule, or an RNA or RNA-containing molecule, or a combination thereof (i.e. DNA-RNA hybrid molecule or complex), comprising or consisting of a double-stranded stem component joined to a single-stranded loop component. The double-stranded stem component comprises a forward strand hybridised by complementary base pairing to a complementary reverse strand, with the 3’ nucleotide of the forward strand joined to the 5’ nucleotide of the single- stranded loop component, and the 5’ nucleotide of the reverse strand joined to the 3’ nucleotide of the single-stranded loop component. The two strands of the stem need not necessarily form a blunt ended structure. There may be additional bases which result in a single stranded overhang and these overhanging bases still provide suitable sites for attachment of detection moieties. The double-stranded stem component may comprise one or more detection moieties, including but not limited to, a fluorophore on one strand (e.g. the forward strand), and one or more quenchers on the opposing strand (e.g. the reverse strand). Other non-limiting examples include a gold or silver nanoparticle on both strands for colorimetric detection, immobilisation of one strand to a gold surface (e.g. the forward strand) and a gold nanoparticle on the opposing strand (e.g. the reverse strand) for SPR detection, and immobilisation of one strand to an electrode surface (e.g. the forward strand) and a methylene blue molecule on the opposing strand (e.g. reverse strand) for electrochemical detection.

[0453] As used herein, the terms “TLM Probe” and / or “TLM Substrate” and / or TLM complex” are used interchangeably to mean a T emperature-dependent Looped Multicomponent probe (TLM), substrate or complex. As used herein, TLM Probes may comprise two or more oligonucleotides. TLM Probes comprising at least two oligonucleotides may have a “first oligonucleotide” and a “second oligonucleotide”. The terms “first oligonucleotide” or “Oligo 1” may be used interchangeably to mean a first oligonucleotide molecule of an TLM probe. The terms “second oligonucleotide” or “Oligo 2” may be used interchangeably to mean a second oligonucleotide molecule of an TLM probe. The terms “third oligonucleotide” or “Oligo 3” may be used interchangeably to mean a third oligonucleotide molecule of an TLM probe. Oligo 1 comprises a “sensor region” that can serve as a substrate for an enzyme only in the presence of the target to be detected. Oligo 1 also comprises a first “capture region” which is complementary to the Oligo 2. Additionally, the Oligo 1 further comprises either a second capture region complementary to a second capture region 2 in Oligo 2, or a second capture region complementary to a third capture region in Oligo 1. When the capture regions 2 and 3 of Oligo 1 hybridised, an internal stem-loop structure may be formed in Oligo 1. Optionally, Oligo 1 may further comprise of an additional capture region which is complementary to a capture region in Oligo 3. Preferably, the capture regions do not overlap with the sensor region. The complementarity regions between Oligo 1 and 2 may be capable of hybridisation or association at temperatures below the melting temperatures of the complementary regions of Oligo 1 and 2. The Oligo 1 and 3 may have regions of complementarity which may be capable of hybridisation or association at temperatures below the melting temperatures of the complementary regions of Oligo 1 and 3.

[0454] Oligo 1 may be connected to a first detection moiety, and Oligo 2 may be connected to a second dye moiety. In some examples, Oligo 1 is directly labelled with a first detection moiety and the Oligo 2 is directly labelled with a second detection moiety. In some examples, Oligo 1 is connected to the first detection moiety via Oligo 3, wherein Oligo 1 further comprises a capture region capable of hybridisation to Oligo 3 by complementary base pairing, and wherein Oligo 3 is directly labelled with the first detection moiety. In such examples, it will be understood that Oligo 1 is nevertheless connected to the first detection moiety (via hybridisation to Oligo 3). Accordingly, Oligo 1 may be connected to the first detection moiety either directly or indirectly.

[0455] As used herein, the term “capture region” refers to a region of an oligonucleotide component capable of hybridising to a capture region of another oligonucleotide or to a capture region of the same oligonucleotide. In an embodiment, the capture region hybridises to the second oligonucleotide component or a region of an oligonucleotide component which hybridises to a second region in the same oligonucleotide component.

[0456] The term “effective Tmintact” or “Tmintact” as used herein refers to the effective melting temperature of the intact TLM probe. Above this temperature, the intact TLM probe dissociates in a way that allows full separation of the fluorophore from the quencher. In an intact probe comprising of complementary capture regions 2 and 3 in Oligo 1, separation of fluorophore from the quencher requires dissociation of capture region 1 between Oligo 1 and Oligo 2 only; therefore, effective Trnmtact is Tm capture region 1. In an intact probe comprising of a second complementary region between Oligo 1 and Oligo 2, both of the complementary regions between Oligo 1 and Oligo 2 need to dissociate for full separation of fluorophore from the quencher, therefore the binding affinity in both regions contributes towards the effective Tm between Oligo 1 and Oligo 2. This means that the effective Trnmtact for this type of probe is combined Tm of the capture regions.

[0457] The term “effective Tmcleaved” or “Tmcleaved” as used herein refers to the effective melting temperature of the cleaved TLM probe. Above this temperature, the cleaved TLM probe dissociates in a way that allows full separation of the fluorophore from the quencher. In a cleaved probe comprising of complementary capture regions 2 and 3 in Oligo 1, separation of fluorophore from the quencher requires Oligo 1 capture regions 2 and 3 to dissociate, therefore the effective Tmcleavedis Tm Oligo 1 capture region 2 / 3 only. In a cleaved probe comprising of two complementary capture regions between Oligo 1 and Oligo 2, the two capture regions in Oligo 1 are separated into the separate fragments; the effective Tmcleavedis Tm complementary region 2 only as only the second Oligo 1 fragment containing capture region 2 needs to dissociate from the probe complex to allow separation of the fluorophore and quencher.

[0458] Oligo 1 may be capable of modification in the presence of a target molecule to be detected. Modification may be mediated by enzymatic activity. The modification by enzymatic activity may be, for example, cleavage by a PlexZyme and / or hydrolysis by the exonuclease activity of a polymerase and / or cleavage by a restriction endonuclease, for example a nicking endonuclease. Some TLM Probes incorporate Oligo Is which comprise nucleic acid enzyme substrates which may be universal, and which are capable of catalytic cleavage by nucleic acid enzymes such as PlexZymes, DNAzymes and aptazymes.

[0459] As used herein the terms “Intact TLM” probe and “unmodified TLM” probe are used interchangeably to refer to an TLM probe wherein the Oligo 1 of the complex has not been modified by cleavage, hydrolysis or nicking by an enzyme. The terms “Cleaved TLM” probe or “Hydrolysed TLM” probe or "modified TLM” probe are used to refer to an TLM probe wherein the Oligo 1 of the probe complex has been modified by cleavage, nicking or hydrolysis by an enzyme in the presence of target. The terms “Intact first oligonucleotide”, “Intact Oligo 1”, “unmodified first oligonucleotide” and “unmodified Oligo 1” are used interchangeably to refer to a first oligonucleotide which has not been modified by cleavage, hydrolysis or nicking by an enzyme. The terms “Cleaved first oligonucleotide”, “Cleaved Oligo 1”, “Hydrolysed first oligonucleotide”, “Hydrolysed Oligo 1”, “modified first oligonucleotide” and “modified Oligo 1” are used to refer to a first oligonucleotide which has been modified by cleavage, hydrolysis or nicking by an enzyme. Modification of a first oligonucleotide can generate multiple “first oligonucleotide fragments”, “Oligo 1 fragments”, “fragments of the first oligonucleotide components” or “fragments of Oligo 1”. Cleavage or hydrolysis of substrates which are present within the first oligonucleotide of an Intact TLM probe complex by an enzyme may generate multiple fragments of the first oligonucleotide which are associated with modified TLM probes.

[0460] As used herein, the term “universal TLM-P probe” refers to a TLM structure which contains a “universal Oligo 1” with a “universal capture region 1”, and a “universal sensor region” which comprises a universal catalytic nucleic acid substrate which can be cleaved by any PlexZyme with complementary substrate binding arms regardless of the sequences of the PlexZyme target sensing arms. The catalytic nucleic acid substrate is not complementary to the target and hence is universal since it may be linked to any target via the incorporation of target specific partzymes. A single universal TLM-P probe can be used as a surrogate marker for any target which is capable of facilitating the cleavage of a specific TLM-P probe. A series of universal TLM-P probes can be incorporated into any multiplex assay designed to analyze any set of targets.

[0461] Additionally, the universal Oligo 1 may be connected to a first detection moiety which may be a quencher molecule that has the capability of quenching a range of different fluorescent detection moieties. In such cases, the universal capture region 1 of Oligo 1 may be capable of hybridisation to any of a series of Oligo 2s, each of which has the same sequence but is labelled with a different fluorescent detection moiety that may be quenched by the quencher connected to Oligo 1.

[0462] Alternatively, a series of Oligo Is for multiplexing or detection of multiple targets may be connected to different first detection moieties, each of which may be a different fluorophore molecule, and each in the series may further comprise a universal capture region 1. In such cases, the universal capture region 1 of Oligo 1 may be capable of hybridisation to a single “universal Oligo 2”, which is labelled with a quencher detection moiety that may quench the multiple fluorophores connected to different Oligo Is.

[0463] Alternatively, or additionally, if Oligo 1 has two capture regions, both of which are universal and capable of hybridisation with a universal Oligo 2 and 3 respectively, then Oligo 1 can be universal with respect to both detection moieties (e.g. the fluorophore and the quencher), as well as with the orientation of these. The sequences of Oligo 2 and 3 could remain the same; however, manufacturers and assay developers would have freedom of choice with respect to which fluorophore and quencher molecules can be connected to Oligo 2 and 3. These could be used in conjunction with Oligo 1 which is not directly labelled with any detection moieties.

[0464] Finally, TLM-P probes comprising nucleic acid enzyme substrates within the sensor regions may be universal with respect to the type of catalytic nucleic acid which can cleave them. By way of example, the same nucleic acid enzyme substrate sequence may be cleaved by PlexZymes, aptazymes, and / or DNAzymes. By way of further example, a nucleic acid enzyme substrate sequence, suitable for cleavage with 10:23 DNAzyme, could also be cleaved by an aptazyme incorporating a 10:23 DNAzyme or by a PlexZyme composed of partzymes harboring partial catalytic core sequences homologous to regions of the 10:23 DNAzyme.

[0465] Various types of TLM Probes are disclosed herein. The term “TLM-P” probe as used herein refers to an TLM complex that may be suitable for cleavage by an PlexZyme assembled in the presence of a target. An TLM-P probe comprising at least two oligonucleotides may have a first oligonucleotide comprising, within its sensor region, a PlexZyme substrate region. The term “TLM-H” probe as used herein refers to an TLM complex that may be suitable for hydrolysis by exonuclease activity, for example, by inherent 5’ to 3’ exonuclease activity of polymerase in the presence of target. An TLM-H probe comprising at least two oligonucleotides may have a first oligonucleotide which comprises, within its sensor region, a sequence which is complementary to the target. The first oligonucleotide may be capable of binding or hybridising to the target at a location which is 3’ of the upstream forward PCR primer. The term “TLM-E” probe as used herein refers to an TLM complex that may be suitable for cleaving or nicking by an exonuclease, for example, by a nicking endonuclease. An TLM-E probe comprising at least two oligonucleotides may have a first oligonucleotide which comprises, within its sensor region, a sequence which is complementary to the target. The first oligonucleotide may be capable of hybridising to the target and forming a double stranded recognition sequence for a specific endonuclease.

[0466] As used herein, the terms “M-Tec Probe” and / or “M-Tec Substrate” and / or M-Tec complex” are used interchangeably to mean a Multiple-component Temperature Controllable probe, substrate or complex. As used herein M-Tec Probes may comprise two or more oligonucleotide components. M-Tec Probes comprising at least two oligonucleotide components may have a “first oligonucleotide component” and a “second oligonucleotide component”. The terms “first oligonucleotide component” or “first component oligonucleotide” or “OC1” may be used interchangeably to mean a first component molecule of an M-Tec probe. The terms “second oligonucleotide component” or “second component oligonucleotide” or “OC2” may be used interchangeably to mean a second component molecule of an M-Tec probe. The first oligonucleotide component comprises a “sensor region” that can serve as a substrate for an enzyme only in the presence of the target to be detected. The first oligonucleotide component also comprises a first “capture region” which is complementary to the second oligonucleotide component. The first oligonucleotide component may be connected to a first detection moiety, and the second oligonucleotide component may be connected to a second dye moiety. The term “Tm OC1 / OC2” as used herein refers to the melting temperature of the complementary regions of the first and second oligonucleotides of an M-Tec Probe.

[0467] The terms “Intact M-Tec” probe and “unmodified M-Tec” probe are used interchangeably to refer to an M-Tec probe wherein the first oligonucleotide component of the complex has not been modified by cleavage, hydrolysis or nicking by an enzyme. The terms “Cleaved M-Tec” probe or “Hydrolysed M-Tec” probe or "modified M-Tec” probe are used to refer to an M-Tec probe wherein the first oligonucleotide component of the probe complex has been modified by cleavage, nicking or hydrolysis by an enzyme in the presence of target.

[0468] As used herein, the term “stem-loop oligonucleotide” will be understood to include “LOCS”, also referred to herein as a “LOCS oligonucleotide”, “LOCS structure”, “LOCS reporter”, “Intact LOCS”, “LOCS probes” and “PlexPlus Probes”. The single-stranded loop component of a LOCS may comprise a region capable of serving as a substrate for a catalytic nucleic acid such as, for example, an MNAzyme (i.e., a PlexZyme), a DNAzyme, a ribozyme, an apta-PlexZyme, or an aptazyme. Additionally, or alternatively, the singlestranded loop component may comprise a region which is complementary to a target nucleic acid (e.g. a target for detection, quantification and the like), and / or amplicons derived therefrom, and which may further be capable of serving as a substrate for an exonuclease enzyme. By way of non-limiting example, the exonuclease may be an inherent activity of a polymerase enzyme. Additionally, or alternatively, the single-stranded loop component region may comprise a region which may: (i) be complementary to the target being detected, (ii) comprise one strand of a double stranded restriction enzyme recognition site; and (iii) be capable of serving as a substrate for a restriction enzyme, for example a nicking endonuclease. As used herein, the terms “split stem-loop oligonucleotide”, “split LOCS”, “split LOCS oligonucleotide”, “split LOCS structure”, “split LOCS reporters”, “split LOCS probes”, “cleaved LOCS” and “degraded LOCS” are used herein interchangeably and will be understood to be a reference to a “LOCS” in which the singlestranded loop component has been cleaved, digested, and / or degraded (e.g. by an enzyme as described herein) such that at least one bond between adjacent nucleotides within the loop is removed, thereby providing a non-contiguous section in the loop region. In split LOCS, the forward and reverse strands of the double-stranded stem portion may retain the ability to hybridise to each other to form a stem in a temperature-dependent manner.

[0469] LOCS are designed to include a cleavable loop region enabling target-dependent cleavage of the loop region by an enzyme generating a split LOCS. This in turn may facilitate detection of the target from a detectable signal generated at specific temperature(s) following association (hybridisation) or dissociation of the stem portion of intact or split LOCS. In contrast, a Molecular Beacon as used herein refers to a stem loop oligonucleotide designed to include a loop region that is not cleavable during the methods described herein. Molecular Beacons may mediate target detection by generating detectable signal at specific temperatures following association (hybridisation) or dissociation (separation) of the loop portion of the probe with the target to be detected. As such, a primary difference between these two types of stem loop structures in the context of the present invention is that LOCS are monitored by measuring changes in signals due to hybridisation or dissociation of the stem region of intact or split LOCS, whereas Molecular Beacons are monitored by measuring changes in signal due to hybridisation or dissociation of the loop region and the target.

[0470] As used herein, the term “universal stem” refers to a double stranded sequence which can be incorporated into any LOCS structure. The same “universal stem” may be used in LOCS which contain Loops which comprise either catalytic nucleic acid substrates or sequence which is complementary to a target of interest. A single universal stem can be used as a surrogate marker for any target which is capable of facilitating the splitting of a specific LOCS. A series of universal stems can be incorporated into a series of LOCS designed for analysis of any set of targets.

[0471] As used herein, the term “universal LOCS” refers to a LOCS structure which contains a “universal stem”, and a “universal Loop” which comprises a universal catalytic nucleic acid substrate which can be cleaved by any PlexZyme with complementary substrate binding arms regardless of the sequences of the PlexZyme target sensing arms. A single universal LOCS can be used as a surrogate marker for any target which is capable of facilitating the splitting of a specific LOCS. A series of universal LOCS can be incorporated into any multiplex assay designed to analyse any set of targets.

[0472] Some LOCS probes comprise nucleic acid enzyme substrates within the loop regions which may be universal, and which are capable of catalytic cleavage by nucleic acid enzymes such as PlexZymes, DNAzymes and aptazymes. Other LOCS probes comprise target specific sequences within the loop region which are capable of catalytic cleavage by protein enzymes including endonucleases and exonucleases. As used herein, the term “universal M-Tec-P probe” refers to a M-Tec structure which contains a first “universal component oligonucleotide” with a first “universal capture region”, and a “universal sensor region” which comprises a universal catalytic nucleic acid substrate which can be cleaved by any PlexZyme with complementary substrate binding arms regardless of the sequences of the PlexZyme target sensing arms. The catalytic nucleic acid substrate is not complementary to the target and hence is universal since it may be linked to any target via the incorporation of target specific partzymes. A single universal M-Tec-P probe can be used as a surrogate marker for any target which is capable of facilitating the cleavage of a specific M-Tec-P probe. A series of universal M-Tec-P probes can be incorporated into any multiplex assay designed to analyze any set of targets.

[0473] Additionally, the first universal oligonucleotide component may be connected to a first detection moiety which may be a quencher molecule that has the capability of quenching a range of different fluorescent detection moieties. In such cases, the first universal capture region of the first oligonucleotide component may be capable of hybridisation to any of a series of second oligonucleotide components, each of which has the same sequence but is labelled with a different fluorescent detection moiety that may be quenched by the quencher connected to the first oligonucleotide component.

[0474] Alternatively, a series of first oligonucleotide components for multiplexing or detection of multiple targets may be connected to different first detection moieties, each of which may be a different fluorophore molecule, and each in the series may further comprise a universal capture region. In such cases, the universal capture region of the first oligonucleotide component may be capable of hybridisation to a single universal second oligonucleotide component, which is labelled with a quencher detection moiety that may quench the multiple fluorophores connected to different first oligonucleotide components.

[0475] Alternatively, or additionally, if the first oligonucleotide component has first and a second capture regions, both of which are universal and capable of hybridisation with universal second and third oligonucleotide components respectively, then the first oligonucleotide component can be universal with respect to both detection moieties (e.g. the fluorophore and the quencher), as well as with the orientation of these. The sequences of the second and third oligonucleotide components could remain the same; however, manufacturers and assay developers would have freedom of choice with respect to which fluorophore and quencher molecules can be connected to the second and third oligonucleotide components. These could be used in conjunction with a first oligonucleotide component which is not directly labelled with any detection moieties. Finally, M-Tec-P probes comprising nucleic acid enzyme substrates within the sensor regions may be universal with respect to the type of catalytic nucleic acid which can cleave them. By way of example, the same nucleic acid enzyme substrate sequence may be cleaved by PlexZymes, aptazymes, and / or DNAzymes. By way of further example, a nucleic acid enzyme substrate sequence, suitable for cleavage with 10:23 DNAzyme, could also be cleaved by an aptazyme incorporating a 10:23 DNAzyme or by a PlexZyme composed of partzymes harboring partial catalytic core sequences homologous to regions of the 10:23 DNAzyme.

[0476] As used herein, the terms “nucleic acid enzyme”, “catalytic nucleic acid”, “nucleic acid with catalytic activity”, and “catalytic nucleic acid enzyme” are used herein interchangeably and shall mean a DNA or DNA-containing molecule or complex, or an RNA or RNA-containing molecule or complex, or a combination thereof (i.e. DNA-RNA hybrid molecule or complex), which may recognise at least one substrate and catalyse a modification (such as cleavage) of the at least one substrate. The nucleotide residues in the catalytic nucleic acids may include the bases A, C, G, T, and U, as well as derivatives and analogues thereof. The terms above include uni -molecular nucleic acid enzymes which may comprise a single DNA or DNA-containing molecule (also known in the art as a “DNA enzyme”, “deoxyribozyme” or “DNAzyme”) or an RNA or RNA-containing molecule (also known in the art as a “ribozyme”) or a combination thereof, being a DNA-RNA hybrid molecule which may recognise at least one substrate and catalyse a modification (such as cleavage) of the at least one substrate. The terms above include nucleic acid enzymes which comprise a DNA or DNA-containing complex or an RNA or RNA-containing complex or a combination thereof, being a DNA-RNA hybrid complex, which may recognise at least one substrate and catalyse a modification (such as cleavage) of the at least one substrate. The terms “nucleic acid enzyme”, “catalytic nucleic acid”, “nucleic acid with catalytic activity”, and “catalytic nucleic acid enzyme” include within their meaning PlexZymes.

[0477] As used herein, the terms “PlexZymes”, “MNAzymes” and “multi-component nucleic acid enzyme” have the same meaning and refer to multi-component nucleic acid enzymes having two or more oligonucleotide sequences (e.g. partzymes) which, only in the presence of an PlexZyme assembly facilitator (for example, a target), form an active nucleic acid enzyme that is capable of catalytically modifying a substrate. The terms PlexZyme”, “MNAzyme” and “multi-component nucleic acid enzyme” comprise bipartite structures, composed of two molecules, or tripartite structures, composed of three nucleic acid molecules, or other multipartite structures, for example those formed by four or more nucleic acid molecules.

[0478] PlexZymes and MNAzymes can catalyse a range of reactions including cleavage of a substrate, and other enzymatic modifications of a substrate or substrates. Component partzymes A and B each bind to an assembly facilitator (e.g. a target DNA or RNA sequence) through base pairing. The PlexZyme only forms when the sensor arms of partzymes A and B hybridise adjacent to each other on the target assembly facilitator. The substrate arms of the PlexZyme engage the substrate, the modification (e.g. cleavage) of which is catalysed by the catalytic core of the PlexZyme, formed by the interaction of the partial catalytic domains of partzymes A and B. PlexZymes may cleave DNA / RNA chimeric reporter substrates.

[0479] It will be understood that the terms “PlexZyme”, “MNAzyme” and “multi-component nucleic acid enzyme” as used herein encompass all known MNAzymes and modified MNAzymes including those disclosed in any one or more of PCT patent publication numbers WO / 2007 / 041774, WO / 2008 / 040095, W02008 / 122084, and related US patent publication numbers 2007-0231810, 2010-0136536, and 2011-0143338 (the contents of each of these documents are incorporated herein by reference in their entirety). Nonlimiting examples of MNAzymes and modified MNAzymes encompassed by the terms “MNAzyme” and “multi-component nucleic acid enzyme” include MNAzymes with cleavage catalytic activity (as exemplified herein), disassembled or partially assembled MNAzymes comprising one or more assembly inhibitors, MNAzymes comprising one or more aptamers (“apta-MNAzymes”), MNAzymes comprising one or more truncated sensor arms and optionally one or more stabilising oligonucleotides, MNAzymes comprising one or more activity inhibitors, multi-component nucleic acid inactive proenzymes (MNAi), each of which is described in detail in one or more of WO / 2007 / 041774, WO / 2008 / 040095, US 2007-0231810, US 2010-0136536, and / or US 2011-0143338.

[0480] As used herein, the terms “partzyme”, “component partzyme” and “partzyme component” refer to a DNA-containing or RNA-containing or DNA-RNA-containing oligonucleotide, two or more of which, only in the presence of an PlexZyme assembly facilitator as herein defined, can together form an “PlexZyme.” In certain preferred embodiments, one or more component partzymes, and preferably at least two, may comprise three regions or domains: a “catalytic” domain, which forms part of the catalytic core that catalyses a modification; a “sensor arm” domain, which may associate with and / or bind to an assembly facilitator; and a “substrate arm” domain, which may associate with and / or bind to a substrate. The terms “sensor arm”, “target sensor arm” or “target sensing arm” or “target arm” may be used interchangeably to describe the domain of the partzymes which binds to the assembly facilitator, for example the target. Partzymes may comprise at least one additional component including but not limited to an aptamer, referred to herein as an “apta-partzyme.” A partzyme may comprise multiple components, including but not limited to, a partzyme component with a truncated sensor arm and a stabilising arm component which stabilizers, the PlexZyme structure by interacting with either an assembly facilitator or a substrate.

[0481] The terms “assembly facilitator”, “PlexZyme assembly facilitator”, “MNAzyme assembly facilitator”, and “target assembly facilitator” as used herein refer to entities that can facilitate the self-assembly of component partzymes to form a catalytically active PlexZyme by interaction with the sensor arms of the PlexZyme. As used herein, assembly facilitators may facilitate the assembly of PlexZymes which have cleavage or other enzymatic activities. In some examples, an assembly facilitator is required for the selfassembly of a PlexZyme. An assembly facilitator may be comprised of a single molecule, or it may be comprised of two or more “assembly facilitator components” that may pair with, or bind to, the sensor arms of one or more oligonucleotide “partzymes”. The assembly facilitator may comprise one or more nucleotide component / s which do not share sequence complementarity with sensor arm / s of the PlexZyme. The assembly facilitator may be a target. The target may be a nucleic acid selected from the group consisting of DNA, methylated DNA, alkylated DNA, RNA, methylated RNA, microRNA, siRNA, shRNA, tRNA, mRNA, snoRNA, stRNA, smRNA, pre- and pri-microRNA, other non-coding RNAs, ribosomal RNA, derivatives thereof, amplicons, or any combination thereof. The nucleic acid may be amplified. The amplification may comprise one or more of: PCR, RT-PCR, SDA, NEAR, HD A, RPA, LAMP, RCA, TMA, RAM, LCR, 3 SR, orNASBA.

[0482] PlexZymes are capable of cleaving substrates incorporated into various probe types, including but not limited to, (i) linear substrates, (ii) substrates which are present within the Loop region of a stem-loop LOCS reporter probe, and (iii) substrates which are present within the first oligonucleotide component of an TLM or an M-Tec-P probe complexes. Linear PlexZyme substrates which are dual labelled are known in the art and have been used for direct detection of nucleic acid sequences and / or for monitoring the accumulation of amplicons by various amplification methods. The cleavage of a linear substrate may separate a fluorophore and quencher allowing detection of a target over a broad range of temperatures. Linear substrates are known in the art as MNAzyme substrates, MNAzyme reporters, MNAzyme probes, PlexZyme substrates, PlexZyme reporters, PlexZyme probes or standard PlexZyme probes. Cleavage of the Loop region of a LOCS by a PlexZyme may generate a Split LOCS structure composed of two fragment which may remain hybridised and associated at temperatures below the melting temperature of the stem and which may separate and dissociate at temperature above the melting temperature of the stem of the split LOCS. Cleavage by a PlexZyme of substrates which are present within the Oligo 1 of an Intact TLM-P probe or the first oligonucleotide component of an Intact M-Tec-P probe complex may generate cleavage fragments of the Oligo 1 or first oligonucleotide component which are associated with cleaved or modified TLM or M-Tec probes.

[0483] PlexZyme cleavage of a substrate may lead to separation of fluorophore and a quencher dye pair, which in turn, may generate a fluorescent signal. Cleavage of various types of PlexZyme substrates may result in changes in fluorescence above background which are observable over a broad range of temperatures, or at only specific temperatures. By way of example, cleavage of dual labelled linear PlexZyme substrates may generate fluorescence above background which may be monitored over a broad temperature range. In other examples, cleavage of substrates within the loop of a LOCS reporter probe may generate fluorescence above background which may only be monitored at temperatures which are above the melting temperature of the cleaved fragments of a Split LOCS structure. In other examples, cleavage of a PlexZyme substrates present within the first oligonucleotide of an TLM-P Probe may generate fluorescence above background which may be monitored at temperatures which are above Tmcleavedand below Tmintact. In yet examples, cleavage of a PlexZyme substrates present within the first oligonucleotide component of an M-Tec-P Probe may generate fluorescence above background which may be monitored at temperatures which are below the melting temperature of the complementary regions of first and second oligonucleotide components.

[0484] The terms “detectable effect” and “detectable signal” are used interchangeably herein and will be understood to have the same meaning. The terms refer to a signal or an effect generated from a detection moiety that is attached to or otherwise associated with an oligonucleotide of the present invention (e.g. a probe, reporter or substrate), typically upon modification of the oligonucleotide to alter its conformation, structure, orientation, position relative to other entit(ies), and the like. The modification may, for example, be induced by the presence of a target that the oligonucleotide is designed to detect. Non-limiting examples of such modifications (e.g. those induced by the presence of the target) include the opening of the stem-loop portion of a Molecular Beacon, the opening of doublestranded portion of Scorpion Uniprobes and Biprobes, the binding of Dual Hybridisation Probes and Double-stranded probes (Yin- Yang probes) to a target sequence, the production of a Catcher Duplex, and cleavage / digestion of a linear PlexZyme substrate or a TaqMan probe, and the like. The detectable signal may be detected by a variety of methods, including fluorescence spectroscopy, surface plasmon resonance (SPR), mass spectroscopy, NMR, electron spin resonance, polarisation fluorescence spectroscopy, circular dichroism, immunoassay, chromatography, radiometry, photometry, scintigraphy, electronic methods, electrochemical methods, UV, visible light or infra-red spectroscopy, enzymatic methods or any combination thereof. The detectable signal / effect can be detected or quantified, and its magnitude may be indicative of the presence and / or quantity of an input such as the amount of a target molecule present in a sample. Further, the magnitude of the detectable signal / effect provided by the detection moiety may be modulated by altering the conditions of a reaction in which an oligonucleotide comprising the detectable moiety is utilised, including but not limited to, the reaction temperature. The capacity of the detection moieties attached to or otherwise associated with the oligonucleotides to generate target-dependent signal, and / or target-independent background signal, can thus be modulated. A detection moiety may be a fluorophore or a quencher.

[0485] As used herein the terms “background signal” and “baseline signal” are used interchangeably and will be understood to have the same meaning. The terms refer to signal generated by a detectable moiety attached to or otherwise associated with an oligonucleotide of the present invention, that is independent of the presence or absence of the specific target which the oligonucleotide is designed to measure or detect under the specific conditions of measurement. As used herein the terms “background fluorescence” and “baseline fluorescence” are used interchangeably and will be understood to have the same meaning. The terms refer to fluorescent signal generated by a detectable moiety attached to or otherwise associated with an oligonucleotide of the present invention, that is independent of the presence or absence of the specific target which the oligonucleotide is designed to measure or detect under the specific conditions of measurement.

[0486] The terms “polynucleotide substrate” and “oligonucleotide substrate” as used herein include any single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases, or analogues, derivatives, variants, fragments or combinations thereof, which is capable of being recognised, acted upon or modified by an enzyme including a catalytic nucleic acid enzyme. A “polynucleotide substrate” or “oligonucleotide substrate” may be modified by various enzymatic activities including but not limited to cleavage. Cleavage or degradation of a “polynucleotide substrate” or “oligonucleotide substrate” may provide a detectable effect for monitoring the catalytic activity of an enzyme. The “polynucleotide substrate” may be cleaved or degraded by one or more enzymes including, but not limited to, catalytic nucleic acid enzymes such as PlexZymes, AptaPlexZymes, DNAzymes, Aptazymes, ribozymes and / or protein enzymes such as exonucleases or endonucleases.

[0487] A “reporter substrate” as used herein is a substrate that is particularly adapted to facilitate measurement of either cleavage or degradation of a substrate or the appearance of a cleaved product in connection with a catalysed reaction. Reporter substrates can be free in solution or bound (or “tethered”), for example, to a surface, or to another molecule. A reporter substrate can be labelled by any of a large variety of means including, for example, fluorophores (with or without one or more additional components, such as quenchers), radioactive labels, biotin (e.g. biotinylation) or chemiluminescent labels.

[0488] As used herein, a “linear PlexZyme substrate” or “linear MNAzyme substrate” is a substrate, for example, a reporter substrate, that is recognised by and acted on catalytically by a plurality of PlexZymes. A “linear PlexZyme substrate” does not contain sequences at its 5’ or 3’ ends which are capable of hybridising to form a stem. Alternatively, PlexZyme substrates may be present within the Loop region of a LOCS probe. In other examples, PlexZyme substrates may be present within the first oligonucleotide component of an M-Tec-P probe or TLM probe.

[0489] As used herein, a “universal substrate” is a substrate, for example, a reporter substrate, that is recognised by and acted on catalytically by a plurality of PlexZymes, each of which can recognise a different assembly facilitator. The use of such substrates facilitates development of separate assays for detection, identification, or quantification of a wide variety of assembly facilitators using structurally related PlexZymes all of which recognise a universal substrate. Further, the same “universal substrate” sequence can be present within multiple probe types including “Linear PlexZyme substrate” and / or the loop region of a “LOCS probe” and / or in the first oligonucleotide component of an “M-Tec-P” probe complex or “TLM” probe complex. These universal substrates can each be independently labelled with one or more labels. In some embodiments, independently detectable labels are used to label one or more universal substrates to allow the creation of a convenient system for independently or simultaneously detecting a variety of assembly facilitators using PlexZymes. In some embodiments the substrates may be capable of catalytic modification by DNAzymes which are catalytically active in the presence of a cofactor, for example a metal ion co-factor such as lead or mercury. In some embodiments, the substrates may be amenable to catalytic modification by aptazymes which may become catalytically active in the presence of an analyte, protein, compound or molecule capable of binding to the aptamer portion of the aptazyme thereby activating the catalytic potential of the nucleic acid enzyme portion.

[0490] The terms “probe” and “reporter” as used herein refer to an oligonucleotide that is used for detection of a target molecule (e.g. a nucleic acid or an analyte). Non-limiting examples of Standard Probes or Reporters, which are well known in the art include, but are not limited to, linear PlexZyme substrates, LOCS probes, M-Tec probes, TaqMan probes or hydrolysis probes, Molecular Beacons, Binary DNA probes (universal Molecular Beacons), Sloppy Beacons, Eclipse probes, Scorpion Uni-Probe, Scorpion Bi-Probes primer / probes, capture / pitcher oligonucleotides, Double-stranded probes (Yin- Yang probes) and dual-hybridisation probes. Embodiments of the present invention combine standard probes with TLM Probes.

[0491] The term “product” refers to the new molecule or molecules that are produced as a result of enzymatic modification of a substrate. As used herein the term “cleavage product” or “cleavage fragment” are used interchangeably to refer to a new molecule produced as a result of cleavage or endonuclease activity by an enzyme. In some embodiments, the products may be produced by enzymatic cleavage or degradation of the first oligonucleotide component of an intactTLM probe, comprising two oligonucleotide fragments, wherein the two oligonucleotide fragments may be capable of either hybridisation or dissociation / separation depending on the temperature of the reaction. In other embodiments, the products may be produced by enzymatic cleavage or degradation of an intact LOCS structure, comprising two oligonucleotide fragments, collectively referred to as a Split LOCS, wherein the two oligonucleotide fragments may be capable of either hybridisation or dissociation / separation depending on the temperature of the reaction. In other embodiments, the products may be produced by enzymatic cleavage or degradation of an intact M-Tec structure, comprising two oligonucleotide fragments, wherein the two oligonucleotide fragments are capable of dissociation / separation.

[0492] As used herein, use of the terms “melting temperature” and “Tm” in the context of a polynucleotide will be understood to be a reference to the melting temperature (Tm) as calculated using the Wallace rule, whereby Tm = 2°C (A+T) + 4°C (G+C) (see Wallace et al., (1979) Nucleic Acids Res. 6, 3543), unless specifically indicated otherwise. The effects of sequence composition on the melting temperature can be understood using the nearest neighbour method, which is governed by the following formula: Tm (°C) = AH° / (AS° + R Infoligo]) - 273.15. In addition to the length and sequence composition of fully or partially complementary regions of sequence, other factors that are known to impact the melting temperature include ionic strength and oligonucleotide concentration. A higher oligonucleotide and / or ion concentration increases the chance of duplex formation which leads to an increase in melting temperature. In contrast, a lower oligonucleotide and / or ion concentration favours dissociation of the stem which leads to a decrease in melting temperature.

[0493] As used herein the term “quencher” includes any molecule that when in close proximity to a fluorophore, takes up emission energy generated by the fluorophore and either dissipates the energy as heat or emits light of a longer wavelength than the emission wavelength of the fluorophore. Non-limiting examples of quenchers include Dabcyl, TAMRA, graphene, FRET fluorophores, ZEN quenchers, ATTO quenchers, Black Hole Quenchers (BHQ), Iowa Black Dark Quenchers and Black Berry Quenchers (BBQ). As used herein, the term “base” when used in the context of a nucleic acid will be understood to have the same meaning as the term “nucleotide”.

[0494] As used herein the term “blocker” or “blocker molecule” refers to any molecule or functional group which can be incorporated into an oligonucleotide to prevent a polymerase using a portion of the oligonucleotide as a template for the synthesis of a complementary strand. By way of a non-limiting example, a hexathylene glycol blocker can be incorporated into, for example, a Scorpion probe to link its 5’ probing sequence to its 3’ priming sequence, wherein the blocker functions to prevent a polymerase using the probing sequence as a template.

[0495] As used herein the terms “normalise”, “normalising” and “normalised”, refer to the conversion of a measured signal (e.g. a detectable signal generated by a detection moiety) to a scale relative to a known and repeatable value or to a control value.

[0496] As used herein, the term “kit” refers to any delivery system for delivering materials. Such delivery systems include systems that allow for the storage, transport, or delivery of reaction reagents (for example labels, reference samples, supporting material, etc. in the appropriate containers) and / or supporting materials (for example, buffers, written instructions for performing an assay etc.) from one location to another. For example, kits may include one or more enclosures, such as boxes, containing the relevant reaction reagents and / or supporting materials. The term “kit” includes both fragmented and combined kits.

[0497] As used herein, the term “fragmented kit” refers to a delivery system comprising two or more separate containers that each contains a subportion of the total kit components. The containers may be delivered to the intended recipient together or separately. Any delivery system comprising two or more separate containers that each contains a subportion of the total kit components are included within the meaning of the term “fragmented kit”.

[0498] As used herein, a “combined kit” refers to a delivery system containing all of the components of a reaction assay in a single container (e.g. in a single box housing each of the desired components).

[0499] It will be understood that use the term “about” herein in reference to a recited numerical value includes the recited numerical value and numerical values within plus or minus ten percent of the recited value or otherwise as understood in the context of the examples described herein.

[0500] It will be understood that use of the term “between” herein when referring to a range of numerical values encompasses the numerical values at each end-point of the range. For example, a polypeptide of between 10 residues and 20 residues in length is inclusive of a polypeptide of 10 residues in length and a polypeptide of 20 residues in length. Any description of prior art documents herein, or statements herein derived from or based on those documents, is not an admission that the documents or derived statements are part of the common general knowledge of the relevant art.

[0501] For the purposes of description all documents referred to herein are hereby incorporated by reference in their entirety unless otherwise stated.

[0502] Abbreviations

[0503] The following abbreviations are used herein and throughout the specification:

[0504] TLM Probe: Temperature dependent Looped Multi-component Probe

[0505] TLM-P Probe: TLM Probe suitable for cleavage by a MNAzyme;

[0506] TEM-H Probe: TLM Probe suitable for hydrolysis by an exonuclease;

[0507] TEM-E Probe: TLM Probe suitable for cleavage by an endonuclease

[0508] Oligo P. First oligonucleotide component of an TLM Probe;

[0509] Oligo 2 Second oligonucleotide component of an TLM Probe;

[0510] Oligo 3: Third oligonucleotide component of an TLM Probe;

[0511] Tricieaved- Melting temperature of a cleaved TLM Probe

[0512] Tmintact: Melting temperature of an intact TLM Probe

[0513] M-Tec Probe: Multi-component Temperature Controlled Probe;

[0514] M-Tec-P Probe: M-Tec Probe suitable for cleavage by a PlexZyme;

[0515] M-Tec-H Probe: M-Tec Probe suitable for hydrolysis by an exonuclease;

[0516] M-Tec-E Probe: M-Tec Probe suitable for cleavage by an endonuclease;

[0517] OOP. First oligonucleotide component of an M-Tec Probe;

[0518] OC2'. Second oligonucleotide component of an M-Tec Probe;

[0519] OC3 -. Third oligonucleotide component of an M-Tec Probe;

[0520] Tm OC1 / OC2. melting temperature of complementary regions of OC1 and OC2 of an M-Tec Probe;

[0521] Tm 0C1 / 0C3. melting temperature of complementary regions of OC1 and OC3 of an M-Tec Probe;

[0522] LOGS: loop connected to stems;

[0523] MNAzyme (also called PlexZyme) '. multi-component nucleic acid enzyme;

[0524] Partzyme: Partial enzyme containing oligonucleotide;

[0525] PCR'. polymerase chain reaction;

[0526] gDNA: genomic DNA;

[0527] NTC: No template control;

[0528] qPCR'. Real-time quantitative PCR; Ct; Threshold cycle;

[0529] Cq; Quantification cycle;

[0530] R2Correlation coefficient;

[0531] nM Nanomolar;

[0532] mM; Millimolar;

[0533] Microlitre;

[0534] / AT; Micromolar;

[0535] dNTP; Deoxyribonucleotide triphosphate;

[0536] NF-H2O: nuclease-free water;

[0537] LN A', locked nucleic acid;

[0538] F fluorophore;

[0539] Q: quencher;

[0540] N= A, C, T, G, or any analogue thereof;

[0541] N’ = any nucleotide complementary to N, or able to base pair with N; (N)x'. any number of N;

[0542] (N’)x'. any number of N’;

[0543] W: A or T;

[0544] R: A, G, or AA;

[0545] rN any ribonucleotide base;

[0546] (rN)x'. any number of rN;

[0547] rR A or G;

[0548] rY: C or U;

[0549] M: A or C;

[0550] H: A, C, or T;

[0551] D: G, A, or T;

[0552] JOE or 6-JOE: 6-carboxy-4',5'-dichloro-2', 7' -dimethoxyfluorescein; FAM or 6-FAM: 6-Carboxyfluorescein;

[0553] HEX'. Hexachlorofluorescein;

[0554] BHQ1'. Black Hole Quencher 1;

[0555] BHQ2: Black Hole Quencher 2;

[0556] RT-PCR: reverse transcription polymerase chain reaction;

[0557] SDA: strand displacement amplification;

[0558] NEAR: Nicking Enzyme Amplification Reaction;

[0559] HDA: helicase dependent amplification;

[0560] RPA: Recombinase Polymerase Amplification;

[0561] LAMP: loop-mediated isothermal amplification; RCA: rolling circle amplification;

[0562] IMA: transcription-mediated amplification;

[0563] 3SR: self-sustained sequence replication;

[0564] NASBA: nucleic acid sequence based amplification;

[0565] LCR: Ligase Chain Reaction;

[0566] RAM: Ramification Amplification Method;

[0567] IB: Iowa Black® FQ;

[0568] IBR: Iowa Black® RQ;

[0569] shRNA: short hairpin RNA;

[0570] siRNA: short interfering RNA;

[0571] mRNA: messenger RNA;

[0572] tRNA: transfer RNA;

[0573] snoRNA: small nucleolar RNA;

[0574] siRNA: small temporal RNA;

[0575] smRNA: small modulatory RNA;

[0576] pre-microRNA: precursor microRNA;

[0577] pri-microRNA: primary microRNA;

[0578] LHS: Left hand side;

[0579] RHS: Right hand side;

[0580] DSO: double stranded oligonucleotide;

[0581] Tm: Melting Temperature;

[0582] RFU: Relative Fluorescence Units;

[0583] CT: Chlamydia trachomatis;

[0584] NG Neisseria gonorrhoeae;

[0585] SPR: surface plasmon resonance;

[0586] GNP: gold nanoparticles;

[0587] DNS: duplex specific nuclease.

[0588] Detailed Description

[0589] The following detailed description conveys exemplary embodiments of the present invention in sufficient detail to enable those of ordinary skill in the art to practice the present invention. Features or limitations of the various embodiments described do not necessarily limit other embodiments of the present invention or the present invention as a whole. Hence, the following detailed description does not limit the scope of the present invention, which is defined only by the claims. The present invention relates to methods and compositions for the multiplexed detection of one or more targets (e.g. nucleic acids, proteins, analytes, compounds, molecules and the like). The methods and compositions each employ a combination of oligonucleotide complexes herein referred to as TLM Probes optionally used together with other oligonucleotide reporters, probes or substrates, which may further be used in combination with various other agent / s.

[0590] Reporters, Probes and Substrates

[0591] According to the present invention, multiplex detection of target molecules is facilitated using TLM Probes in combination with another nucleic acid suitable for use as a probe in a multiplex detection assay.

[0592] Many nucleic acid probes for detection of nucleic acid targets have been described and are well known in the art. Suitable nucleic acid probes that can be used in combination with TLM Probes include, but are not limited to, LOCS probes, M-tec probes, linear PlexZyme substrates, TaqMan or Hydrolysis probes, Molecular Beacons, Binary DNA probes (universal Molecular Beacons) Sloppy Beacons, Eclipse probes, Amplifluor / Sunrise primer probes, Scorpion Uni-Probe, Scorpion Bi-Probes, dual-hybridisation probes, Double-stranded probes, (Yin-Yang probes) and Capture / Pitcher probes

[0593] In some embodiments, these nucleic acid probes bind directly to the target or target amplicon to facilitate their detection, however, probe types that incorporate PlexZyme substrates, and Capture / Pitcher oligonucleotides, provide an exception as they may be universal and suitable for detection of any target.

[0594] In some embodiments, the nucleic acid probes generate fluorescence in the presence of target due to enzymatically mediated cleavage or degradation, for example, TLM probes, M-Tec Probes, LOCS Probes, linear PlexZyme substrates and TaqMan or Hydrolysis probes.

[0595] In other embodiments, the nucleic acid probes provide different levels of fluorescent signal as a result of a conformation change induced by binding to a target or target amplicon (e.g. Molecular Beacons, Binary DNA probes (universal Molecular Beacons), Sloppy Beacons, Eclipse probes, Scorpion Uni-Probe, Scorpion Bi-Probes, Double-stranded probes (Yin-Yang probes) and dual-hybridisation probes).

[0596] In the TOCE system, the Catcher changes fluorescence as a result of conformation changes induced by binding and extension of the Pitcher which is only activated and released in the presence of target.

[0597] Any, or all, of these types of reporter nucleic acid probes are suitable for use in conjunction with TLM Probes to mediate detection of multiple targets by measurement of changes related to a single detection moiety, including but not limited to, a change in fluorescence measured at a single wavelength.

[0598] Oligonucleotides for TLM Probes or for probes used in combination with TLM Probes can be synthesised according to standard protocols. For example, they may be synthesised by phosphoramidite chemistry, using nucleoside and non-nucleoside phosphoramidites in sequential synthetic cycles that involves removal of the protective group, coupling the phosphoramidites, capping and oxidation, either in solid-phase or solution-phase and optionally in an automated synthesiser device. Alternatively, they may be purchased from commercial sources. Non-limiting examples of commercial sources from which linear, LOCS or M-Tec PlexZyme substrates, TaqMan or Hydrolysis probes, Molecular Beacons, Binary DNA probes (universal Molecular Beacons), Sloppy Beacons, Eclipse probes, Scorpion Uni-Probe, Scorpion Bi-Probes, dual-hybridisation probes, Double-stranded probes (Yin- Yang probes) and Capture / Pitcher probes can be purchased or otherwise obtained include: PlexZyme substrates can be purchased from SpeeDx (plexpcr.com); TaqMan and hydrolysis probes can be purchased from Thermo Fisher Scientific (www.thermofisher.com), Sigma Aldrich (www.sigmaaldrich.com), Promega (www.promega.com), Generi Biotech (www.generi-biotech.com); Molecular Beacons, Binary DNA probes (universal Molecular Beacons) and Sloppy beacons may be purchased from Integrated DNA Technologies (www.idtdna.com), Eurofins (www.eurofmsgenomics.com), Sigma Aldrich (www.sigmaaldrich.com) and TriLink BioTechnologies (www.trilinkbiotech.com); Eclipse probes can be purchased from Integrated DNA Technologies (www.idtdna.com); Scorpion Uni-Probes can be purchased from Sigma Aldrich (www.sigmaaldrich.com) and Bio-Synthesis (https: / / www.biosyn.com); Scorpion bi-probes can be purchased from Bio-Synthesis (https: / / www.biosyn.com); Dual-hybridisation probes can be purchased from BioSynthesis (https: / / www.biosyn.com), Sigma Aldrich (www.sigmaaldrich.com) and Eurofins (www.eurofmsgenomics.com); Double-stranded probes (Yin-Yang probes) can be purchased from Integrated DNA Technologies (www.idtdna.com); and Catcher Pitcher assays may be purchased from Seegene (www.seegene.com).

[0599] TLM Probe Oligonucleotides

[0600] Provided herein are methods and compositions which extend the capacity for multiplex analysis of nucleic acid targets. These methods and compositions employ Temperature-dependent Looped Multi-component Probes herein referred to as TLM Probes. By way of example, TLM Probes are multiple-component complexes comprising at least two oligonucleotides wherein a first oligonucleotide (Oligo 1) is labelled with a first detection moiety and is capable of being modified by enzymatic activity in the presence of a target, and a second oligonucleotide (Oligo 2) is labelled with a second detection moiety.

[0601] The main function of this probe is to produce a target-dependent signal at a predetermined temperature range; while no signal is produced outside of this temperature range regardless of presence or absence of target. A key feature of this probe is that the effective binding affinity between probe components is different in intact and modified probes. The distance between the F and Q is different in intact and cleaved probes, as is the case with all PlexProbes. In the presence of a target, a sensor region of the probe is modified, for example by cleavage or hydrolysis by an enzyme. The effective binding affinity is lowered once the probe is cleaved by enzymatic modification. This in turn lowers the effective melting temperature (Tm) of the modified probe i.e. temperature that allows full separation of the fluorophore from the quencher. This means that an unmodified (intact) probe has a higher effective Tm compared to a modified (cleaved) probe. Therefore, at temperatures above Tmcleavedand below Tmintact the intact probes are non-fluorescent due to close proximity of the detection moieties and cleaved probes are fluorescent due to separation of the detection moieties, allowing discrimination between presence and absence of target. At temperatures below Tmcleavedboth intact and cleaved probes are non-fluorescent (quenched) due to close proximity of the detection moieties, producing a constant level of background fluorescence. At temperatures above Tmintact, both intact and cleaved probes will dissociate, which allows separation of the detection moieties and constant fluorescence. Therefore, the probe is capable of producing a target-dependent signal at a confined temperature range. Within the probe structure, Oligo 1 comprises a first capture region capable of hybridisation to the first capture region of Oligo 2 by complementary base pairing to form a double-stranded portion. Oligo 1 also comprises of a sensor region of Oligo 1 that is capable of being modified by enzymatic activity. Oligo 1 is connected to a detection moiety by direct labelling, or via an additional region of complementarity with a third oligonucleotide (Oligo 3) which is labelled with the detection moiety. Oligo 2 is also labelled with a second detection moiety. The first and second detection moiety may be, for example, a fluorophore and a quencher or vice versa. In some embodiments, Oligo 1 or 2 may be indirectly labelled.

[0602] In addition to the common features described above, Oligo 1 may comprise another capture region. Depending on which other region this capture region is complementary to, two distinct exemplary structures can be distinguished. These are described in the next two paragraphs. The first exemplary structure (Figure 1) further comprises capture region 2 complementary to capture region 3 in Oligo 1. When the capture regions 2 and 3 are hybridised, an internal stem-loop structure is formed in Oligo 1. The binding affinity between the capture regions 2 and 3 depends on whether the Oligo 1 is intact or cleaved (Figure 1 (i) and (ii)). In intact probes, the hybridised capture regions 2 and 3 form an internal stem-loop structure; in cleaved probes, the capture regions 2 and 3 are separated into different Oligo 1 fragments. Since intramolecular bonds are stronger than intermolecular bonds, the Tm of the stem-loop structure in intact probes is higher than the Tm of capture region 2 / 3 of the cleaved probes. In a cleaved probe, separation of fluorophore from the quencher requires Oligo 1 capture regions 2 and 3 only to dissociate, therefore the effective Tmcleavedis Tm Oligo 1 capture region 2 / 3 only. In an intact probe, separation of fluorophore from the quencher requires dissociation of capture region 1 between Oligo 1 and Oligo 2 only; therefore, Tmintact is Tm capture region 1. In an embodiment, in an intact probe, Tm capture region 1 is higher than Tm capture region 2 / 3. As the effective Tmintact is higher than Tmcleaved, a fluorescence signal is generated at temperatures above Tmcleavedand below Tmintact in the presence of target only.

[0603] The second exemplary structure (Figure 2) further comprises of a second complementary region between Oligo 1 and Oligo 2. This second double-stranded region is formed by the second capture region of Oligo 1 hybridising to the second capture region of the Oligo 2 by complementary base pairing. Therefore, there are two complementary regions between Oligo 1 and Oligo 2; enzymatic cleavage of Oligo 1 occurs between the two regions. When the probe is intact, both of these complementary regions need to dissociate for full separation of fluorophore from the quencher, therefore the binding affinity in both regions contributes towards the effective Tm between Oligo 1 and Oligo 2 (Figure 2 (i) and (ii). The first complementary region increases the Tm of the second complementary region and vice versa, due to the increased binding affinity between Oligo 1 and Oligo 2 when there are two complementary regions between them. This means that the effective Tmintact for this type of probe is combined Tm of the capture regions. In a cleaved probe, the two capture regions in Oligo 1 are separated into the separate fragments; the Tm of the two complementary regions is now independent from each other, and the effective Tmcleavedis Tm complementary region 2 only as only the second Oligo 1 fragment containing capture region 2 needs to dissociate from the probe complex to allow separation of the fluorophore and quencher. Preferably, Tm capture region 1 is higher than Tm capture region 2. As the effective Tmintact is higher than Tmcleaved, a fluorescence signal is generated at temperatures above Tmcleavedand below Tmintact in the presence of target only. If present, a third oligonucleotide component (Oligo 3) is capable of hybridisation with the Oligo 1 via complementary capture regions in Oligo 1 and Oligo 3. The Tm of this capture region should be above effective Tmintact, to ensure that the Oligo 1 and Oligo 3 remain hybridised at temperatures below effective Tmintact.

[0604] When all oligonucleotide components are hybridised, and the Oligo 1 is unmodified, the fluorophore and quencher are in close proximity resulting in intact TLM Probe complexes which are quenched. In the presence of a target, a sensor region of the Oligo 1 is modified, for example by cleavage or hydrolysis by an enzyme. Enzymatic modification of the sensor region of the Oligo 1 generates a first fragment comprising of the capture region 1 and a second fragment comprising of capture region 2and the first detection moiety. At temperatures below the effective Tmcleaved, the probe remains fully hybridised even when Oligo 1 has been modified by the presence of target, keeping the fluorophore and quencher in close proximity. This results in a quenched TLM probe regardless of presence or absence of target. Increasing the temperature over the effective Tmcleavedenables the second fragment of Oligo 1 to dissociate from the probe complex, therefore spatially separating the first and second detection moieties and generating a detectable signal. Resultant target-dependent increases in fluorescence can be measured at temperatures above effective Tmcleavedand below effective Tmintact. At temperatures above the Tmintact, all the probe components dissociate, and the dye moieties are separated, resulting in background levels of fluorescence which are constant regardless of the presence or absence of target. At this temperature the Oligo 2 will no longer hybridise to either the unmodified Oligo 1 in reactions where no target is present, or to a hydrolysed / cleaved fragment of the Oligo 1, which has been modified in the presence of target.

[0605] In this manner TLM Probes will generate target dependent increases in fluorescence at temperatures above effective Tmcleavedand below effective Tmintact but no change in fluorescence will be observed regardless of the presence or absence of target at temperatures below effective Tmcleavedand above Tmintact.

[0606] Various types of TLM Probes are disclosed and exemplified (see e.g., Figures 1-4, Figure 24-27). Although exemplified with a fluor ophore / quencher pair, the skilled addressee will recognise that any other suitable detection moiety(-ies) may be used for the same purpose. One type of TLM Probe is suitable for modification / cleavage by an PlexZyme in the presence of target. These probes are herein referred to as TLM-P probes and an exemplary components and complexes are illustrated in Figure 1, 2,3 and 4. An TLM-P may have two oligonucleotides, namely a first oligonucleotide (Oligo 1) labelled at one terminus with a quencher and comprising a sensor region that serves as a substrate for a PlexZyme, and a second oligonucleotide (Oligo 2) labelled with a fluorophore (Figures 4 (i) and (iii)). Alternatively, TLM complexes may be labelled with a fluorophore at one terminus and comprise a sensor region that serves as a substrate for a PlexZyme, and an Oligo 2 labelled with a quencher (Figure 4 (ii) and (iv)). The Oligo 1 comprises a first capture region capable of hybridisation to the first capture region of Oligo 2; these regions are capable of hybridisation or association at temperatures below the effective Tmintact. Additionally, Oligo 1 may comprise of a second capture region complementary to a second capture region in Oligo 2 (Figure 2, 4(iii) and (iv)); alternatively, Oligo 1 may comprise of capture region 2 and 3 that form an internal stem-loop structure (Figure 1, 4(i) and (ii)).

[0607] TLM probes are designed to be cleavable by PlexZymes which can assemble from component partzymes, when the partzymes bind adjacently to complementary regions on the target to be detected (Figures 3-5). The TLM may then bind to the PlexZyme following hybridisation of the Oligo 1 with the substrate binding arms of a PlexZyme. In some embodiments the Oligo 2 hybridises to the capture region 1 of the Oligo 1 which does not hybridise / bind to the substrate binding arms of the partzymes as illustrated in several figures e.g. Figure 3 and Figure 4. Cleavage of the sensor region of the Oligo 1 of the TLM-P results in generation of two Oligo 1 fragments, one of which contains the quencher and capture region 2 and other of which contains the capture region 1 and capture region 3 if present (Figure l(ii) and Figure 2(ii)). When Oligo 1 comprises of capture regions 2 and 3, these two regions are in different Oligo 1 fragments after cleavage of the sensor region. Since intramolecular bonds are stronger than intermolecular bonds, the hybridised Oligo 1 capture regions 2 and 3 of the intact TLM probes will generally melt at higher temperatures than the hybridised Oligo 1 capture regions 2 and 3 of the split, cleaved or degraded TLM probes. At “middle” temperatures above the effective Tmcleavedand below the effective Tmintact, an increase in fluorescence is indicative of the presence of the target which facilitated the assembly of the PlexZyme (Figure 5(ii) and Figure 6(ii)). In the absence of target, TLM probes are not cleaved and there is no increase in fluorescence above background during the reaction at this temperature (Figure 5(v) and Figure 6(v)). At “low” temperatures below the effective Tmcleaved, no change in fluorescence is observed regardless of the presence or absence of the target (Figure 5(i) and (iv) and Figure 6(i) and (iv)).

[0608] Background fluorescence from quenched TLM probe is constant and is not affected by the presence or absence of target. At “high” temperatures above the effective Tmintact, no change in fluorescence is observed regardless of the presence or absence of the target (Figure 5(iii) and (vi) and Figure 6(iii) and (vi)). Background or baseline fluorescence resulting from dissociation of Oligo 1 and Oligo 2 is constant and is not affected by the presence or absence of target. In some embodiments the TLM-P probe may be universal. A universal TLM-P structure may contain a universal Oligo 2 and a universal Oligo 1 comprising a universal sensor region which includes a universal catalytic nucleic acid substrate which can be cleaved by any PlexZyme with complementary substrate binding arms regardless of the sequences of the PlexZyme target sensing arms. A single universal TLM-P probe can be used as a surrogate marker for any target which is capable of facilitating the cleavage of a specific TLM-P probe. A series of universal TLM-P probes can be incorporated into any multiplex assay designed to analyse any set of targets. Additionally, the universal Oligo 1 may be connected to a first detection moiety which may be a fluorophore, and the universal Oligo 2 may be connected to a second detection moiety which may be a quencher moiety that has the capability of quenching a range of different fluorophores. In such cases the Oligo 2 may have a universal capture region 1 may be capable of hybridisation to a universal capture region 1 in a series of Oligo Is, each of which has a different sensor region and is labelled with a different fluorophore that may be quenched by the quencher connected to the universal Oligo 2. Some TLM-P probes comprise nucleic acid enzyme substrates within the sensor region which may be universal, and which are capable of catalytic cleavage by nucleic acid enzymes such as PlexZymes, DNAzymes, aptazymes and ribozymes. By way of example, the same universal TLM-Probe may be used in conjunction with many target specific PlexZymes to detect a range of nucleic acid targets and further may be used in conjunction with many target specific aptazymes to detect a range nonnuclei c acid targets.

[0609] Another type of TLM Probe is suitable for hydrolysis by exonuclease activity, for example, by Taq polymerase in the presence of target. These probes are herein referred to as TLM-H probes and an exemplary components and complexes are illustrated in Figure 24A, Figure 25-26 and Figure 27A. An TLM-H may have two oligonucleotide components, the Oligo 1 and Oligo 2. The Oligo 1 may be labelled with a quencher at one terminus and have a sensor region, at least a portion of which is complementary to the target to be detected; whilst the Oligo 2 may be labelled with a fluorophore. Alternatively TLM-H complexes may comprise an Oligo 1 labelled with a fluorophore at one terminus, and an Oligo 2 labelled with a quencher. Capture region 1 in each of the probe components Oligo 1 and Oligo 2 are complementary and are capable of hybridisation or association at temperatures below the effective Tmintact (Figures 25(i) and (iv). Additionally, Oligo 1 may comprise of a second capture region complementary to a second capture region in Oligo 2; alternatively, Oligo 1 may comprise of complementary capture regions 2 and 3 that form a stem-loop structure. In some embodiments, the Oligo 2 hybridises to a capture region of the Oligo 1 which does not hybridise / bind to the target. TLM-H probes are designed to be hydrolysed by exonuclease activity in the presence of target. During PCR, at least a portion of the sensor region of the Oligo 1 of the TLM-H may bind to the target amplicon and at least a portion of the sensor region may be hydrolysed by the exonuclease activity of polymerase. Hydrolysis of the Oligo 1 of the TLM-H Probe results in generation two Oligo 1 fragments, one of which contains the quencher and capture region 2 and other of which contains the capture region 1 and capture region 3 if present (Figure 25). When Oligo 1 comprises of capture regions 2 and 3, these two regions are in different Oligo 1 fragments after cleavage of the sensor region. Since intramolecular bonds are stronger than interm olecular bonds, the hybridised Oligo 1 capture regions 2 and 3 of the intact TLM probes will generally melt at higher temperatures than the hybridised Oligo 1 capture regions 2 and 3 of the split, cleaved or degraded TLM probes. At “middle” temperatures above the Tmcleavedand below the Tm intact, an increase in fluorescence is indicative of the presence of the target (Figure 25(ii)). In the absence of target, TLM-H probes are not hydrolysed and there is no increase in fluorescence above background during the reaction (Figure 25 (v)) at this temperature. At “low” temperatures below the effective Tmcleaved, no change in fluorescence is observed regardless of the presence or absence of the target (Figure 25(i) and (iv)). Background fluorescence from quenched TLM probe is constant and is not affected by the presence or absence of target. At “high” temperatures above the effective Tm intact, no change in fluorescence is observed regardless of the presence or absence of the target (Figures 25(iii) and (vi)).

[0610] Background fluorescence resulting from dissociation of Oligo 1 and Oligo 2 is constant and is not affected by the presence or absence of target.

[0611] In some embodiments, TLM Probes are used in combination with hairpin universal PlexZyme probes, also known as LOCS (Loops Connected to Stems) Probes (Figure 7(ii), Figure 8, Figure 10-11) and / or universal multicomponent PlexZyme probes known as M-Tec Probes (Figure 7(iii) and Figure 8). In other embodiments TLM Probes can be combined with other Probe and Substrate types known in the art, including, but not limited to, linear PlexZyme substrates, standard dual -labelled TaqMan probes or Hydrolysis probes Molecular Beacons, Binary DNA probes (universal Molecular Beacons), Sloppy Beacons, Eclipse probes, Scorpion Uni-Probes or Bi-Probes, Capture / Pitcher Oligonucleotides, Double-stranded probes (Yin-Yang probes), and dual-hybridisation probes.

[0612] The combination of TLM Probes with other Probe or Substrate types allows greater multiplexing capacity, wherein multiple targets can be detected, identified and / or quantified at a single wavelength. By way of example, an TLM Probe, together with a LOCS probe and a M-Tec Probe, all of which incorporate the same detection moiety (e.g. the same fluorophore) can be used to individually discriminate multiple targets within a single reaction. Alternatively, multiple targets may be discriminated within a single reaction using an TLM Probe, an M-Tec Probe and a LOCS probe, which incorporate different detection moi eties that emit signal at a similar wavelength that may monitored in the same fluorescent channel on a detection instrument. The approach involves measurement of the signal generated from the probes at discrete temperatures. In some embodiments, a first target is measured at a first temperature by monitoring changes in fluorescence associated with modification of an M-Tec Probe, a second target is measured at a second temperature by monitoring changes in fluorescence associated with modification of a TLM probe, and a third target is measured at a third temperature by monitoring changes in fluorescence associated with modification of a LOCS probe.

[0613] The properties of components of the TLM probes, and other probe types with which they may be combined to increase multiplexing capacity, is part of the design of the multiplexed assay. In particular, the Tm of regions of complementarity between components of probes (intermolecular bonds), or within probes (intramolecular bonds) and between components or probes and the targets (intermolecular bonds) influences the capacity to combine various probe types. Exploitation of these properties of the probes and / or their components, allows manipulation of the association or dissociation of regions of probes, and probe complex components, at defined temperatures. In turn, these properties influence whether or not various probe types generate target specific fluorescence at specific acquisition temperatures or whether only background fluorescence is observed in the presence or absence of target. This may be better understood by consideration of the scenarios for different probe types tabulated in Tables 1, 2 and 3 below. The scenarios are exemplary only and one skilled in the art will appreciate that there are many other scenarios that can be envisaged for combining probes to allow analysis of multiple targets at a single wavelength when the principles of manipulation of target dependent fluorescence and background outlined above and below are applied. Table 1: Relation of Tm of components at acquisition Temperature 1 (T emp 1), acquisition Temperature 2 (Temp 2) and acquisition Temperature 3 (Temp 3) in the presence of target (+ T) or absence of target (-T) when Temp 1 < Temp 2 < Temp 3. Scenarios which result in increased detectable signal which is measured as Fluorescence above background (F) or Background Signal only (B) at Temp 1, 2 and 3 are tabulated. _ _

[0614] Probe Type Interrelationship between the Tm’s of Temp 1 Temp 2 Temp 3 probes and components and Temp 1, 2 and

[0615] 3

[0616] TLM Probe Effective Tmcleaved> Temp 1 + / - T = B + T = F + / - T = B Effective Tmcleaved< Temp 2 - T = B Effective Tmintact > Temp 2

[0617] Effective Tmintact < Temp 3

[0618] M-Tec Probe Tm 0Cl / 0C2 > Temp 1 + T = F + / - T = B + / - T = B Tm OC1 / OC2 < Temp 2 - T = B

[0619] Tm OC1 / OC2 < Temp 3

[0620] LOCS probe Intact Tm Stem > Temp 3 + / - T = B + / - T = B + T = F Split Tm Stem > Temp 2 - T = B Split Tm Stem < Temp 3

[0621] Molecular Tm Stem < Tm Loop / Target + T = F + / - T = B + / - T = B Beacon Tm Stem and Tm Loop / Target > Temp 1 - T = B

[0622] Tm Stem and Tm Loop / Target < Temp 2

[0623] Binary Probe Tm Stem < Tm Loop / Connector + T = F + / - T = B + / - T = B (Universal oligonucleotides - T = B

[0624] Molecular Tm Stem and Tm Loop / Connector

[0625] Beacon) oligonucleotides > Temp 1

[0626] Tm Stem and Tm Loop / Connector

[0627] oligonucleotides < Temp 2

[0628] Tm Stem and Tm Loop / Connector

[0629] oligonucleotides < Temp 3

[0630] Linear Not Applicable + T = F + T = F + T = F PlexZyme - T = B - T = B - T = B probe

[0631] TOCE Probe Tm Catcher / Pitcher > Temp 1 + T = F + / - T = B + / - T = B Tm Catcher / Pitcher < Temp 2 - T = B

[0632] Tm Catcher / Pitcher > Temp 1 + T = F + T = F + / - T = B

[0633]

[0634] Tm Catcher / Pitcher > Temp 2 - T = B - T = B Tm Catcher / Pitcher < Temp 3

[0635] Tm Catcher / Pitcher > Temp 1 + T = F + T = F + T = F Tm Catcher / Pitcher > Temp 2 - T = B - T = B - T = B

[0636]

[0637] Tm Catcher / Pitcher > Temp 3

[0638] As such, the following features can be used to manipulate generation of target dependent fluorescence at specific acquisition temperatures for various probe types. By way of example, when the effective Tmcleavedof an TLM Probe is above the first ‘low’ acquisition temperature and Tmintact is below the third ‘high’ acquisition temperature, target dependent fluorescence will be observed only at the second ‘middle’ acquisition temperature. In the absence of target, the probe will only generate background fluoresnce at the second temperature, as this temperature is below Tmintact. At first and third temperatures, background fluorescence only will be observed regardless of the presence or absence of target.

[0639] When the components of M-Tec probe have a Tm OC1 / OC2 which is above a first acquisition temperature but below the second acquisition temperature, target dependent fluorescence will be observed only at the first acquisition temperature. Background fluorescence only will be observed at the first temperature in the absence of target, and background fluorescence only will be observed at the second and third temperature regardless of the presence or absence of target.

[0640] When the Tm of the stem of an intact / uncleaved LOCS probe is above the third acquisition temperature, and the Tm of the stem of a cleaved split LOCS probe is above the second acquisition temperature but below the third acquisition temperature, target dependent fluorescence will be observed only at the third acquisition temperature.

[0641] Background fluorescence only will be observed at the third temperature in the absence of target and background fluorescence only will be observed at the first and second temperature regardless of the presence or absence of target.

[0642] When the Tm of the stem of a Molecular Beacon is less than the Tm of the loop / target hybrid, and the Tm of both the stem and the loop / target hybrid are above the first acquisition temperature but below the second acquisition temperature, target dependent fluorescence will be observed only at the first acquisition temperature.

[0643] Background fluorescence only will be observed at the first temperature in the absence of target and background fluorescence only will be observed at the second and third temperature regardless of the presence or absence of target.

[0644] Linear PlexZyme probes which have been cleaved in the presence of a target do not have the same capacity to have the fluorescence controlled by temperature. As such, once these probes are cleaved in the presence of target, they will fluoresce at the first, second and third acquisition temperatures. These probes will produce background signal only when no target is present.

[0645] In the presence of a target, the TOCE system produces double stranded Catcher / Pitcher complexes specific for each target. These complexes can be designed to be of any length and hence have Tm’s at various temperatures set at the designer’s discretion. If the Tm of the Catcher / Pitcher complex is above the first acquisition temperature but below the second acquisition temperature, target dependent fluorescence will be observed only at the first acquisition temperature. Background fluorescence only will be observed at the first temperature in the absence of target and at the second and third temperature regardless of the presence or absence of target. If the Tm of the Capture / Pitcher complex is above the second acquisition temperature, target dependent fluorescence will be observed at both the first and second acquisition temperatures.

[0646] Background fluorescence only will be observed at the first and second temperatures in the absence of target and at the third temperature regardless of the presence or absence of target. If the Tm of the Capture / Pitcher complex is above the third acquisition temperature, target dependent fluorescence will be observed at the first, second and third acquisition temperatures. Background fluorescence only will be observed at the first, second and third temperatures in the absence of target.

[0647] The capacity to manipulate target dependent fluorescence and background at various temperatures for specific probe types, as exemplified above, provides a broad general approach for designing systems for combining probe types that allow detection of targets at specific temperatures only. Further, it provides a wide range of options for detecting multiple targets at a single wavelength. This may be illustrated by non-exhaustive exemplary combinations tabulated in Table 2. Table 2: Generation of Fluorescence (F) above background or Background only (B) when probe types are combined to detect Target 1 (Tl), Target 2 (T2) and Target 3 (T3) at a single wavelength under temperature relationships consistent with those defined in Table 1 and, where indicated, as further defined within Table 2. The presence and absence of Target are indicated as plus (+) or minus (-) respectively. Each pair of probe types in the table can be labelled with the same fluorophore and read at the same wavelength. Alternatively, each pair of probe types in the table can be labelled with different fluorophores provided the fluorophores have similar wavelengths which can be read in the same fluorescent channel.

[0648] Probes (Target detected) Temp 1 Temp 2 Temp 3 Analysis M-Tec Probe (Tl) + Tl = F + / - T1 = B + / - T1 =B Tl = F at Temp 1 TLM Probe (T2) + / - T2 = B + T2 = F + / - T2 = B T2 = F at Temp 2 LOCS Probe (T3) + / - T3 = B + / - T3 = B + T3 = F T3 = F at Temp 3 Molecular Beacon (Tl) + Tl = F + / - T1 = B + / - T1 =B Tl = F at Temp 1 TLM Probe (T2) + / - T2 = B + T2 = F + / - T2 = B T2 = F at Temp 2 LOCS Probe (T3) + / - T3 = B + / - T3 = B + T3 = F T3 = F at Temp 3 Molecular Beacon (Tl) + Tl = F + / - T1 = B + / - T1 =B Tl = F at Temp 1 TLM Probe 1 (T2) + / - T2 = B + T2 = F + / - T2 = B T2 = F at Temp 2 TLM Probe 2 (T3) + / - T3 = B + / - T3 = B + T3 = F T3 = F at Temp 3 TLM Probe 1 (Tl) + Tl = F + / - T1 = B + / - T1 =B Tl = F at Temp 1 TLM Probe 2 (T2) + / - T2 = B + T2 = F + / - T2 = B T2 = F at Temp 2

[0649]

[0650] TLM Probe 3 (T3) + / - T3 = B + / - T3 = B + T3 = F T3 = F at Temp 3

[0651] As exemplified in Table 2 there are numerous ways in which specific probe types can be combined to analyse multiple targets at a single wavelength. The relationships between the Tm of intramolecular bonds for each probe type, and for intermolecular bonds between probe components and probes and targets, as exemplified in Table 1, underpin the capacity to multiplex different probe types for this purpose when signal is acquired at specific temperatures. By way of example, an TLM Probe designed to detect Target 2 can be combined with an M-Tec Probe designed to detect Target 1 and a LOCS probe designed to detect Target 3. In this scenario an increase in fluorescence above background will be observed for Target 1 at the first acquisition temperature only, an increase in fluorescence above background will be observed for Target 2 at the second acquisition temperature only, and an increase in fluorescence above background will be observed for Target 3 at the third acquisition temperature only. Conversely, background signal only will be observed at the first acquisition temperature in the absence of for Target 1 regardless of the presence or absence of Target 2 and 3; background signal only will be observed at the second acquisition temperature in the absence of Target 2 regardless of the presence or absence of Target 1 and 3; and background signal only will be observed at the third acquisition temperature in the absence of Target 3 regardless of the presence or absence of Target 1 and 2. As such the presence of Target 1, and / or determination of the number of copies, can be determined by analysis of data acquired at temperature 1; the presence of Target 2, and / or determination of the number of copies, can be determined by analysis of data acquired at temperature 2; and the presence of Target 3, and / or determination of the number of copies, can be determined by analysis of data acquired at temperature 3.

[0652] A Molecular Beacon designed to detect Target 1 can be combined with an TLM and a LOCS probe designed to detect Target 2 and 3. In this scenario, an increase in fluorescence above background will be observed for Target 1 at the first acquisition temperature only, an increase in fluorescence above background will be observed for Target 2 at the second acquisition temperature only, and an increase in fluorescence above background will be observed for Target 3 at the third acquisition temperature only. Conversely, background signal only will be observed at the first acquisition temperature in the absence of for Target 1 regardless of the presence or absence of Target 2 and 3; background signal only will be observed at the second acquisition temperature in the absence of Target 2 regardless of the presence or absence of Target 1 and 3; and background signal only will be observed at the third acquisition temperature in the absence of Target 3 regardless of the presence or absence of Target 1 and 2. As such the presence of Target 1, and / or determination of the number of copies, can be determined by analysis of data acquired at temperature 1; the presence of Target 2, and / or determination of the number of copies, can be determined by analysis of data acquired at temperature 2; and the presence of Target 3, and / or determination of the number of copies, can be determined by analysis of data acquired at temperature 3.

[0653] Multiple TLM Probe may be combined with other probe types for multiplex detection. By way of example, two TLM Probe designed to detect Target 2 and 3 can be combined with an M-Tec Probe designed to detect Target 1. Alternatively, three TLM Probes designed to detect Targets 1, 2 and 3 can be combined. In both of these scenarios an increase in fluorescence above background will be observed for Target 1 at the first acquisition temperature only, an increase in fluorescence above background will be observed for Target 2 at the second acquisition temperature only, and an increase in fluorescence above background will be observed for Target 3 at the third acquisition temperature only. Conversely, background signal only will be observed at the first acquisition temperature in the absence of for Target 1 regardless of the presence or absence of Target 2 and 3; background signal only will be observed at the second acquisition temperature in the absence of Target 2 regardless of the presence or absence of Target 1 and 3; and background signal only will be observed at the third acquisition temperature in the absence of Target 3 regardless of the presence or absence of Target 1 and 2. As such the presence of Target 1, and / or determination of the number of copies, can be determined by analysis of data acquired at temperature 1; the presence of Target 2, and / or determination of the number of copies, can be determined by analysis of data acquired at temperature 2; and the presence of Target 3, and / or determination of the number of copies, can be determined by analysis of data acquired at temperature 3.

[0654] In addition to the above, there are several probe combinations allowing more than three acquisition temperatures at a single wavelength. As demonstrated above, the TLM probe can be designed to be detected at any ‘middle’ temperature while only producing a background signal at all other temperatures. Theoretically, this allows an infinite number of TLM probes to be combined for multiplex detection. Furthermore, any number of TLM probes may be combined with other probe types for multiplex detection. For example, two TLM probes may be combined with a M-Tec and a LOCS Probe for detection of four targets, or three TLM probes may be combined with a M-Tec and a LOCS Probe for detection of five targets.

[0655] One skilled in the art will appreciate that there are many other scenarios that can be envisaged for combining probes to allow analysis of multiple targets at a single wavelength when the principles of manipulation of target dependent fluorescence and background outlined above are applied.

[0656] Table 2 gives multiple options for combinations of various probe types which incorporate the principle described in Table 1. It is possible to combine these further by using different combinations of different probes labelled with the same fluorophores to be read in separate channels. This may be further understood by the non-exhaustive scenarios tabulated in Table 3.

[0657] Table 3: Exemplary reactions formats wherein multiple probe types, labelled with multiple Fluorophores, for example Fl, F2 or F3, are combined to develop highly multiplex reactions whereby one or more Targets, for example Tl, T2, T3, T4, T5, T6, T7, T8, T9, T10, Til, T12 are measured by a single F with acquisition of fluorescence at multiple temperatures, for example Temp 1, Temp 2, Temp 3 and Temp 4. When relationship of the Tm of component with respect to each other, and the acquisition temperature are as defined in Tables 1 and 2 then Fluorescence (F) above background, or Background only (B), could be measured as tabulated below.

[0658] Fluorophore Probes (Target Temp 1 Temp 2 Temp 3 Temp 4 detected)

[0659] Fl M-Tec Probe + Tl = F + / - T1 = B + / - T1 =B + / - T1 =B (Tl) + / - T2 = B + T2 = F + / - T2 = B + / - T2 = B TLM Probe (T2) + / - T3 = B + / - T3 = B + T3 = F + / - T3 = B TLM Probe (T3) + / - T4 = B + / - T4 = B + / - T4 = B + T4 = F LOCS Probe (T4)

[0660] F2 Molecular + T5 = F + / - T5 = B + / - T5 = B + / - T5 = B Beacon (T5) + / - T6 = B + T6 = F + / - T6 = B + / - T6 = B TLM Probe (T6) + / - T7 = B + / - T7 = B + T7 = F + / - T7 = B TLM Probe (T7) + / - T8 = B + / - T8 = B + / - T8 = B + T8 = F LOCS Probe (T8)

[0661] F3 Molecular + T9 = F + / - T9 = B + / - T9 = B + / - T9 = B Beacon (T9) + / - T10 = B + T10 = F + / - T10 = B + / - T10 = B TLM Probe (T10) + / - T11 = B + / - T11 =B + Til = F + / - T11 = B TLM Probe (Til) + / - T12 = B + / - T12 = B + / - T12 = B + T12 = F LOCS Probe

[0662]

[0663] (T12)

[0664] The scenarios outlined in Table 3 provide an exemplary strategy which would allow for the detection, specific identification and / or quantification of twelve targets present in a single reaction when fluorescence is acquired at four temperatures at three different wavelengths specific for three fluorophores. At a first wavelength, specific for a first fluorophore, a first target could be monitored at a first acquisition temperature using an M- Tec probe specific for Target 1, second and third targets could be monitored at a second and third acquisition temperature using two TLM probe specific for Target 2 and 3, and a fourth target could be monitored at a fourth acquisition temperature using a LOCS probe specific for Target 4. At a second wavelength, specific for a second fluorophore, a fifth target could be monitored at a first acquisition temperature using a Molecular Beacon specific for Target 5, sixth and seventh targets could be monitored at a second and third acquisition temperature using two TLM probe specific for Target 6 and 7 and an eight target could be monitored at a fourth acquisition temperature using a LOCS probe specific for Target 8. At a third wavelength, specific for a third fluorophore, a ninth target could be monitored at a first acquisition temperature using a Molecular Beacon specific for Target 9, tenth and eleventh targets could be monitored at a second and third acquisition temperature using two TLM probe specific for Target 10 and 11 and a twelfth target could be monitored at a fourth acquisition temperature using a LOCS probe specific for Target 12.

[0665] In all of the exemplary strategies outlined in Tables 1, 2 and 3, and the discussion relating to these, the TLM Probe included in the mix could be either an TLM Probe or an TLM-H Probe or an TLM- E probe since all types of exhibit similar properties with respect to their components and capacity to generate target dependent fluorescence at a ‘middle’ acquisition temperature and background only at a Tow’ and ‘high’ acquisition temperature, provided the effective Tm requirements are met.

[0666] A strategy for combining an TLM Probe with an M-tec and LOCS probe to analyse three targets at a single wavelength may be further understood by the illustration in Figure 8. Figures 8A and 8B schematically illustrate an approach for multiplex analysis of three targets using the combination of one M-Tec-P Probe (A), one TLM Probe (B) and one LOCS probe (C) all of which are labelled with the same fluorophore (F) and quencher (Q). Reaction mixes contain an intact M-Tec-P Probe (Ai), an intact TLM Probe (Bi) and an intact LOCS probe (Ci). In the presence of Target 1 (Tl), PlexZyme 1 (Pl) assembles and cleaves the sensor region in the intact M-Tec-P Probe to generate a cleaved M-Tec-P Probe (Ac). In the presence of Target 2 (T2), PlexZyme 2 (P2) assembles and cleaves the intact TLM probe to generate a cleaved, split TLM Probe (Be). In the presence of Target 3 (T3), PlexZyme 3 (P3) assembles and cleaves the intact LOCS probe to generate a cleaved, Split LOCS Probe (Cc). Figure 8B Panel (i) illustrates structures which can form at acquisition temperature 1 which is below the Tm OC1 / OC2 of both intact and cleaved M-Tec Probes, below the effective Tmcleavedof the TLM Probe, and below the Tm of the stem of both intact and Split LOCS probe. Figure 8B Panel (ii) illustrates structures which can be formed at acquisition temperature 2 which is above the Tm OC1 / OC2 of both intact and cleaved M-Tec-P probes, above Tmcleavedand below Tmintact of TLM Probe, and below the Tm of the stem of both intact and Split LOCS probe. Figure 8B Panel (iii) illustrates structures which can be formed at acquisition temperature 3 which is above the Tm OC1 / OC2 of both intact and cleaved M-Tec-P probes, above Tmintact of TLM Probe, and above the Tm of the stem of the Split LOCS probe but below the Tm of the stem of the intact LOCS probe. At temperature 1 fluorescence above background would be generated in the presence of Target 1 but not in the absence of Target 1. At this temperature the TLM and LOCS probes may be cleaved but no fluorescence above background would be generated from the resultant Split LOCS or TLM probes since both probes remain fully hybridised and the fluorophore would remain quenched. Hence an increase in fluorescence above background at temperature 1 would indicate the presence of Target 1 and background fluorescence would be the same regardless of the presence or absence of Target 2 and 3. At temperature 2, the first fragment of the cleaved TLM probe will dissociate, therefore separating the fluorophore from the quencher and producing a fluorescence signal above background. The intact TLM probe remains fully hybridised, therefore only producing background fluorescence. At this temperature the LOCS probe may be cleaved but no fluorescence above background would be generated from the resultant Split LOCS probes since it remains fully hybridised and the fluorophore would remain quenched. The first and second oligonucleotide components of both cleaved and intact M-Tec probe complexes would dissociate and only generate background fluorescence. Hence an increase in fluorescence above background at temperature 2 would indicate the presence of Target 2 and background fluorescence would be the same regardless of the presence or absence of Target 1 and 3. At temperature 3, both the intact and cleaved TLM Probes dissociate and generate background fluorescence only. Similarly, both cleaved and intact M-Tec-P probes would dissociate and only generate background fluorescence. At this temperature the stem of the Split LOCS probe, but not intact LOCS probe, would dissociate resulting in an increase in Fluorescence above background. Hence an increase in Fluorescence above background at temperature 3 would indicate the presence of Target 3 and background fluorescence would be the same regardless of the presence or absence of Target 1 and 2.

[0667] A strategy for combining an TLM-H Probe with a M-Tec and LOCS probe to analyze three targets at a single wavelength may be further understood by the illustration in Figure 26. This figure illustrates an approach for multiplex analysis of three targets using the combination of one M-Tec-P Probe (A), one TLM-H Probe (B) and one LOCS probe (C) all of which are labelled with the same fluorophore (F) and quencher (Q). Reaction mixes contain an intact M-Tec-P Probe (Ai), an intact TLM-H Probe (Bi) and an intact LOCS probe (Ci). In the presence of Target 1 (Tl), PlexZyme 1 (Pl) assembles and cleaves the sensor region in the intact M-Tec-P Probe to generate a cleaved M-Tec-P Probe (Ac). In the presence of Target 2 (T2), the 5 '-3' exonuclease activity of polymerase hydrolyses the sensor region in the intact TLM-H probe to generate a cleaved, split TLM-H Probe (Be). In the presence of Target 3 (T3), PlexZyme 3 (P3) assembles and cleaves the intact LOCS probe to generate a cleaved, Split LOCS Probe (Cc). Multiplex analysis of signals generated by these three probes at different temperatures follows the same principle as illustrated in Figure 8B.

[0668] Persons skilled in the art will recognise that TLM probes may detect the target directly or they may detect target amplicons following amplification of the target using methods well known in the art. Further, persons skilled in the art will recognise that TLM probes may detect amplicons in real time or at the end of the reaction. By the way of example, a melt curve analysis could be used at the end of an amplification reaction containing TLM probes to determine the presence or absence of the specific target and / or its amplicon. Intact TLM probes remain quenched at temperatures below effective Tmintact, and produce fluorescence once Oligo land Oligo 2 dissociate at temperatures above effective Tmintact.

[0669] At the melting temperature, where 50% of the TLM probes are dissociated, an observable peak would appear in the first derivative of the melting curve. Cleaved TLM probes on the other hand produce fluorescence at a lower temperatures than an intact TLM probe, due to Tmintact being lower than Tmcleaved, and therefore there is an observable peak at a lower temperature in the first derivative of the melting curve. Therefore, the presence of the melt peak at a higher temperature would indicate the absence of the target, and the presence of the melt peak at a lower temperature would indicate the presence of the target.

[0670] An TLM probe may be used in combination with one or more probes for a single channel multiplexing, which could be analysed with melt curve analysis. Multiple TLM probes with different effective Tmcleavedand Tmintact would produce differentiable melt peaks at each Tmcleavedand Tmintact in the presence or absence of each target in the first derivative of the melting curve. Alternatively, an TLM probe may be used with other probes that produce signals in a temperature-dependent manner, as these probes produce melt peaks in the first derivative of the melting curve, which may only appear either in the presence or absence of the target. For example, a LOCS probe may produce a peak at a specific low temperature in the presence of the specific target, and at a specific higher temperature in the absence of the target. Similarly, for example, a M-Tec probe may produce a peak at a specific temperature in the absence of target, and no peak would be observed at that temperature in the presence of target. The analysis of the peaks produced by either intact or cleaved LOCS or M-Tec probes could be done simultaneously with the analysis of the peaks produced by TLM probes. Persons skilled in art will recognise that any possible combination of probe types could be used where more than two probes are used for analysis per fluorescent channel or wavelength, if each probe used in the reaction produce peaks that are differentiable in the melt curve analysis.

[0671] LOCS Oligonucleotides

[0672] Exemplary LOCS oligonucleotides for use in the present invention are illustrated in Figure 10. The exemplary Intact LOCS oligonucleotide shown (Figure 10A, LHS) has a Loop region, a Stem region and a fluorophore (F) / quencher (Q) dye pair. Although exemplified with a fluorophore / quench er pair, the skilled addressee will recognise that any other suitable detection moiety(-ies) may be used for the same purpose. The Loop region contains a substrate region which is amenable to enzymatic cleavage or degradation in the presence of target or target amplicons. Cleavage or degradation of the Loop within an Intact LOCS generates the Split LOCS duplex (Figure 10B, RHS).

[0673] In some embodiments, the melting temperature (“Tm”) of the Intact LOCS oligonucleotide is higher than the Tm of the Split LOCS structure.

[0674] Since intramolecular bonds are stronger than intermolecular bonds, the stem regions of the intact LOCS structures will generally melt at a higher temperature than the stems of the Split, cleaved or degraded LOCS oligonucleotide structures. For example, the Stem of intact LOCS A will melt at Tm A which is higher than Tm B which is the temperature at which Split LOCS stem melts (Figure 10B). The presence of fluorescence at a temperature which allows melting of Split LOCS but not Intact LOCS is indicative of the presence of target, or target amplicons. In the exemplary LOCS depicted in Figure 11, the sequence of the Loop region of a LOCS oligonucleotide may be, for example, a substrate for a PlexZyme or other catalytic nucleic acid / s.

[0675] An exemplary LOCS suitable for use in the invention, may contain a Loop region comprising a substrate for a catalytic nucleic acid as illustrated in Figure 11. In these embodiments, LOCS oligonucleotides may comprise universal substrates which can be used to detect any target. The LOCS oligonucleotide contains a stem region, a fluorophore quencher / dye pair (alternative detection moiety(-ies) as described herein may be employed) and an intervening Loop region which comprises a universal substrate for a catalytic nucleic acid such as an PlexZyme. The PlexZyme may detect a target directly or may be used to detect amplicons generated during target amplification. The PlexZyme forms when the target sensor arms of the partzymes each hybridise to a target, or to target amplicons, by complementary base pairing to form the active catalytic core of the PlexZyme. The Loop region of the LOCS oligonucleotide hybridises to the substrate binding arms of the PlexZyme by complementary base pairing and the substrate within the Loop is cleaved by the PlexZyme. This generates a Split LOCS structure which has a stem with a Tm B that is lower than the Tm A of the Intact LOCS. Measurement of a fluorescent signal at temperatures above Tm B but below Tm A is indicative of the presence of target in the reaction. Persons skilled in the art will recognise that the targets can be detected in real time or at the end of the reaction.

[0676] Other mechanism for degradation of the loop region of the intact LOCS probes are known in the art. Alternative designs for LOCS probes have loop regions which can directly bind to the target. These types of LOCS probes can be degradaded by exonuclease activity of a polymerase or by a restriction endonuclease. It will be understood that the terms “LOCS” probe as used herein encompass all known LOCS probes including those disclosed in the PCT patent publication numbers WO 2020 / 031156 Al and W02020206509A1 (the contents of each of these documents are incorporated herein by reference in their entirety).

[0677] Further Exemplary Embodiments

[0678] In certain embodiments, reporter oligonucleotides including TLM Probes of the present invention may be used to detect a target directly without being coupled to a target amplification protocol. In other exemplary embodiments reporter probes or substrates may be used to detect target amplicons generated by target amplification technologies including, but not limited to, PCR, RT-PCR, SDA, NEAR, HD A, RPA, LAMP, RCA, TMA, 3 SR, LCR, RAM or NASB A. Cleavage or degradation of an TLM probe may occur in real time during target amplification or may be performed following amplification, at the end point of the reaction. Oligo 1 may be modified by target-dependent cleavage or degradation mediated by the enzymatic activity of a catalytic nucleic acid including, but not limited to a PlexZyme, an aptazyme, an apta-MNAzyme, a DNAzyme, a ribozyme, or by the enzymatic activity of a protein enzyme including an exonuclease or an endonuclease. By way of non-limiting example, the exonuclease activity may be an inherent catalytic activity of, for example, a polymerase. By way of non-limiting example, the endonuclease activity may be an inherent catalytic activity of, for example, a restriction enzyme including a Nicking endonuclease, a riboendonuclease or a duplex specific nuclease (DSN).

[0679] Reactions of the present invention may detect multiple targets simultaneously using a single TLM probe or probes in combination with other type(s) of reporter probes. As would be evident to persons skilled in the art, standard reporter probes can further be combined with additional TLM probes, for example, wherein each TLM probe may comprise a different universal substrate within its Oligo 1.

[0680] The reaction mix may further comprise additional reporter probes or substrates combined with TLM probes labelled with different fluorophore and quencher pairs. By way of non-limiting example, a Reporter oligonucleotide 1 and TLM probe 2 may be labelled with fluorophore A, and Reporter oligonucleotide 3 and TLM probe 4 may be labelled with fluorophore B. Target-dependent fluorescence associated Fluorophore A may be detected at multiple temperatures in Fluorescence channel A and target-dependent fluorescence associated Fluorophore B may be detected at multiple temperatures in fluorescence channel B on an instrument. The reaction mix may further comprise at least one pair of reporter probes comprising a reporter probe or substrate and a TLM probe labelled with different fluorophores A and B which emit fluorescence at a similar wavelength which can be monitored in a single fluorescent channel on an instrument. In a further exemplary embodiment, a TLM probe may be combined with a M-Tec Probe and a LOCS Probe wherein all contain the same fluorophore / quencher dye pair and the substrate regions are specific for a DNAzyme or a ribozyme, for example, a DNAzyme or ribozyme which can only be catalytically active in the presence of a specific metal ion. Specific DNAzymes and ribozymes are known in the art to require a metal cation cofactor to enable catalytic activity. For example, some DNAzymes and ribozymes can only be catalytically active in the presence of, for example, cadmium, lead or mercury. Such metals may be present in, for example, an environmental sample. A reaction could include one TLM probe suitable for cleavage by a DNAzyme, which is, for example, mercury dependent, wherein the presence of mercury in a sample could result in cleavage of the substrate in the Oligo 1 and generation of a fluorescent signal. The same reaction could also include a M-Tec reporter which contains a loop comprising a substrate for a DNAzyme, which is, for example, cadmium dependent, wherein the presence of cadmium in a sample could result in cleavage of the M-Tec Probe and generation of a fluorescent signal at a temperature below the effective Tmcleavedof the TLM probe. The same reaction could also include a LOCS reporter which contains a loop comprising a substrate for a DNAzyme, which is, for example, lead dependent, wherein the presence of lead in a sample could result in cleavage of the LOCS and generation of a fluorescent signal at a temperature higher than the effective Tmintact of the TLM Probe. An increase in fluorescence at a first temperature, which is below the Tm OC1 / OC2 of M-Tec Probe, effective Tmcleavedof TLM Probe and below the Tm of the Split LOCS, would indicate the presence of cadmium. An increase in fluorescence at a second temperature, which is above the Tm OC1 / OC2 of M-Tec Probe, effective Tmcleavedof TLM Probe and the Tm of the Split LOCS, would indicate the presence of mercury. An increase in fluorescence at a third temperature, which is above the Tm OC1 / OC2 of M-Tec Probe, effective Tmintact of TLM Probe and the Tm of the Split LOCS but below the Tm of Intact LOCS, would indicate the presence of lead. One skilled in the art would readily recognise that multiple probes cleavable in the presence of specific metal cofactors, could be combined in a single reaction and detected either in real time or at the end of the reaction.

[0681] Non-limiting examples of target nucleic acids (i.e. polynucleotides), which may be detected using TLM probes in combination with other well-known probes types could include DNA, methylated DNA, alkylated DNA, complementary DNA (cDNA), RNA, methylated RNA, microRNA, siRNA, shRNA, mRNA, tRNA, snoRNA, stRNA, smRNA, pre- and pri-microRNA, other non-coding RNAs, ribosomal RNA, derivatives thereof, amplicons thereof or any combination thereof (including mixed polymers of deoxyribonucleotide and ribonucleotide bases). TLM probes could also be used to detect proteins or other molecules which can be recognised by aptamers incorporated into aptazymes or apta-MMAzymes. The catalytic activity of aptazymes or apta-MMAzyme may be inhibited in the absence of target molecules that are capable of binding to the aptamer domain of these nucleic acid enzymes. Binding of a molecule, for example a protein, to the aptamer domain could activate the catalytic potential of an aptazyme or apta-MMAzyme which could facilitate cleavage of a first oligonucleotide component of an TLM probe.

[0682] Generation of Detectable Signals

[0683] The methods and compositions of the present invention utilise detection moieties to provide detectable signals. The nature of the detectable signal that the moieties are capable of producing will depend on the type of detection moiety and / or the conformation of the oligonucleotide to which it is associated.

[0684] Any suitable detection moiety can be utilised that is capable of providing a detectable signal upon the modification of an oligonucleotide to which it is associated. Non-limiting examples of suitable detection moieties include fluorophores for fluorescent signal generation, nanoparticles for colorimetric or SPR signal generation, reactive moieties (e.g. alkaline phosphatase or peroxidase enzymes) for chemiluminescent signal generation, electroactive species for electrochemical signal generation, and any combination thereof. By way of non-limiting example, suitable electroactive species include Methylene blue, Toluene Blue, OracetBlue, ferrocene, Hoechst 33258, [Ru(phen)3]2+ orDaunomycin and the most common electrode materials include gold, glassy carbon, pencil graphite or carbon ionic liquid, Methods for the detection and measurement of fluorescent, chemiluminescent, colorimetric, surface plasmon resonance (SPR) and electrochemical signals are well known to persons skilled in the art.

[0685] By way of non-limiting example, oligonucleotides of the present invention, including TLM probes, may have one or more fluorophores attached. The detectable signal inherently generated by the fluorophore may be quenched due to proximity to one or more quencher molecules. For example, and without limitation, the fluorophore(s) may be attached to a single strand of a double-stranded stem portion (e.g. at the 5' or 3' terminus) of a Molecular Beacon or a LOCS, and the quencher(s) may be attached to an opposing strand of the double-stranded stem portion (e.g. at the 5' or 3' terminus). Alternatively, the quencher(s) may be attached to another entity (e.g. a surface or another oligonucleotide) to which the oligonucleotide is bound such that the detectable signal inherently generated by the fluorophore may be quenched. In the presence of a target, the oligonucleotide may undergo a modification that distances the fluorophore(s) from the quencher molecule(s) thus generating a detectable signal.

[0686] Additionally, or alternatively, the oligonucleotides (including Oligo 1 or Oligo 2) may be attached to gold nanoparticles (GNP) for colorimetric detection. When GNPs- are aggregated in close proximity to each other they exhibit a purple colour (i.e. absorbance at a longer wavelength) and when GNPs are separated they exhibit a red colour (i.e. absorbance at a shorter wavelength) wherein, a measurable colour change from purple to red (e.g. TLM, M-Tec, LOCS, linear PlexZyme substrates, Catcher-Pitcher probes, TaqMan probes and restriction enzyme probes) or alternatively from red to purple (e.g. dual hybridisation probes) is indicative of the presence of a specific target in a sample.

[0687] Additionally, or alternatively, the oligonucleotides (including Oligo 1 or Oligo 2) and / or oligonucleotide components may be attached to a GNP and / or a gold surface for SPR detection of a target in a sample. When GNPs move into close proximity, or alternatively when they move away from a gold surface, they can generate a change in measurable SPR signal where a decrease in SPR signal using some approaches (e.g. TLM Probes, M-Tec Probes, LOCS, linear PlexZyme substrates, TaqMan probes and restriction enzyme probes) can be indicative of the presence of a specific target in a sample or alternatively wherein an increase in SPR signal using other approaches (e.g. Catcher-Pitcher probes and dual hybridisation probes) can be indicative of the presence of a specific target in a sample.

[0688] Additionally, or alternatively, the oligonucleotide reporter and probes (including Oligo 1 or Oligo 2) and / or oligonucleotide components may be attached to electroactive species and / or on an electrode surface for electrochemical detection. When the oligonucleotides attached to electroactive species move into close proximity with, or alternatively when they move away from, an electrode surface they can generate a measurable change in oxidation or reduction current. In some embodiments (e.g. TLM Probes, M-Tec Probes, LOCS, linear PlexZyme substrates, TaqMan probes and restriction enzyme probes), the resulting measurable signal arising from an electroactive species moving away from the electrode surface is indicative of the presence of a specific target in a sample. Alternatively, in other embodiments (e.g. Catcher-Pitcher probes and dual hybridisation probes), the resulting measurable signal arising from an electroactive species moving into close proximity to the electrode surface is indicative of the presence of a specific target in a sample.

[0689] In some embodiments, the compositions and methods of the present invention utilise TLM oligonucleotide components attached to a specific detection moiety in combination with another oligonucleotide probe that is attached to the same detection moiety, or a similar detection moiety that generates a detectable signal capable of being detected simultaneously with signal generated by the detectable moiety of the TLM probe (e.g. using a single type of detector such as one fluorescence channel, or a specific mode of colorimetric, surface plasmon resonance (SPR), chemiluminescent, or electrochemical detection).

[0690] Analyses of Fluorescent Signals

[0691] Without limitation and by way of example only, detectable moieties used in accordance with the present invention include fluorescent signals generated by these detectable moieties upon modification, cleavage or digestion of oligonucleotide probes to which they are attached, coupled, or otherwise associated, including dissociation or association of oligonucleotide components, or fragments of oligonucleotide components, of TLM Probe complexes, can be analysed in any suitable manner to detect, differentiate, and / or quantify target molecules in accordance with the methods of the present invention.

[0692] By way of non-limiting example, measurements of fluorescent signal at a single temperature, or at multiple temperatures, may be obtained at various time points within a reaction suitable for detecting modification of TLM probe oligonucleotides. By way of nonlimiting examples, these time points may comprise (i) a time point at, or near, the initiation of a reaction, and / or (ii) a single time point, or multiple time points, during the course of the reaction; and / or (iii) a time point at the conclusion or end-point of the reaction.

[0693] In some embodiments, measurement of fluorescent signal may be obtained at three or more temperatures at each cycle during an amplification reaction, such as during PCR amplification. Analysis may be performed by comparing levels of fluorescence obtained at a first, second and / or third temperature and / or at a further temperature.

[0694] In several embodiments, measurement of fluorescent signal may be obtained at three temperatures in reactions which are tailored to measure three targets at the same wavelength. In other embodiments the measurement of fluorescent signal may be obtained at four temperatures in reactions which are tailored to measure four targets at the same wavelength.

[0695] In some embodiments a first target may be detected using an TLM probe where fluorescent signals can be measured at multiple time points, or at multiple cycles, for example, at each cycle during PCR. In the same reaction additional targets may be detected using a M-Tec or LOCS by comparing pre-PCR and post-PCR fluorescence levels. In such embodiments, quantitative data may be determined for the first target, whilst qualitative data may be generated for the additional targets. By way of non-limiting example, the first TLM Probe may be an PlexZyme substrate cleaved by a first PlexZyme in the presence of a first target and monitored in real time; whereas a LOCS and / or M-tec probe may be cleaved by a second / third PlexZyme in the presence of a second or third target and monitored using end-point detection analysis.

[0696] In some embodiments an increase in fluorescence at the first temperature is indictive of the presence of the first or second or third target, an increase in fluorescence at the second temperature is indictive of the presence of the second or third target and an increase in fluorescence at the third temperature is indictive of the presence of the third target and so forth. In other embodiments an increase in fluorescence at the first temperature is indictive of the presence of the first target, an increase in fluorescence at the second temperature is indictive of the presence of the second target, an increase in fluorescence at the third temperature is indictive of the presence of the third target and so forth. In other embodiments, measurement of fluorescent signal may be obtained at two or more temperatures at each cycle during PCR, and amplification curves may be plotted for each series of measurement obtained at each temperature. Threshold fluorescence values can be assigned to each amplification plot for each specific temperature and Ct or Cq values may be measured as the cycle number where the amplification plots cross the threshold values.

[0697] In embodiments wherein a first probe is an TLM probe for detection of target 1 and the second probe is a Linear PlexZyme Probe for detection of target 2; and wherein measurement of fluorescent signal is obtained at two temperatures at each cycle during PCR, the total fluorescent signal from the TLM probe and linear PlexZyme substrate is measured at the lower temperature, which is above effective Tmcleavedand below effective Tmintact, and the detectable signal measured at a higher temperature, which is above effective Tm intact of the TLM Probe, is solely from the linear PlexZyme substrate, the TLM probe signal can be calculated using the differential between the signal measured at the two temperatures, and the Cq value for target 1 can be determined thereafter; and the Cq value for target 2 can be determined from the signal measured at the higher temperature.

[0698] In other embodiments, measurement of fluorescent signal may be obtained at two or more temperatures at each cycle during PCR, and amplification curves may be plotted for each series of measurement obtained at each temperature. Threshold fluorescence values can be assigned to each amplification plot for each specific temperature and Cq values may be measured as the cycle number where the amplification plots cross the threshold values. In embodiments wherein a first probe for detection of a first target is an TLM probe, is combined with a second probe for detection of a second target, which may be a M-Tec reporter, and a third probe for detection of a third target, which may be a LOCS reporter; and wherein measurement of fluorescent signal is obtained at three temperatures at each cycle during PCR, the Cq measured using fluorescent signal from the TLM Probe at the ‘middle’ temperature, which is above effective Tmcleavedand below the effective Tm intact of the TLM Probe and above Tm OC1 / OC2 of M-Tec Probe and below the Tm of a Split LOCS, may allow direct quantification of the starting concentration of a first target; and the Cq measured using fluorescent signal from the M-Tec reporter at the Tow’ temperature, which is below effective Tmcleavedof the TLM Probe and Tm OC1 / OC2 of M-Tec Probe and the Tm of a Split LOCS, may allow direct quantification of the starting concentration of a second target, and the Cq measured using fluorescent signal from the LOCS reporter at the ‘high’ temperature, which is above the Tm OC1 / OC2 of the M-Tec Probe and effective Tmintact of TLM Probe and above Tm of the Split LOCS but below the Tm of the Intact LOCS, may allow direct quantification of the starting concentration of a third target.

[0699] By way of non-limiting example, baseline fluorescence signal can be obtained by measuring fluorescence at selected temperatures, for example a first, second and third temperature, at a time point which is either at, or near, the initiation of a reaction, for example pre-PCR. The probes included may comprise of for example an TLM Probe, a M-Tec Probe, a LOCS Probe, a linear PlexZyme substrate or a TaqMan probe or a Molecular Beacon,. Analysis may be performed by comparing levels of fluorescence obtained at the first and second temperature at a time point at the initiation of a reaction (e.g. pre-PCR) and levels of fluorescence obtained at the first and second temperatures at a time point, or time points, during and / or after the reaction (e.g. during PCR or post-PCR).

[0700] Exemplary applications of TLM probes when combined with other reporter / probes Detection of targets during or following Target amplification

[0701] TLM probes of the present invention may be used to determine the presence of amplified target nucleic acid sequences. No particular limitation exists in relation to amplification techniques to which the M-Tec probes may be applied. Amplicons generated by various reactions may be detected by M-Tec probes, provided the presence of target amplicons can promote the cleavage or degradation of an Intact TLM probe to produce cleaved TLM probes. Non-limiting examples of methods useful in cleaving or degrading the sensor region of the first oligonucleotide components of TLM probes include cleavage by PlexZymes, DNAzymes, aptazymes, Apta-MNAzymes, ribozymes, restriction enzymes, endonucleases or degradation by exonucleases including but not limited to the exonuclease activity of a polymerase.

[0702] In general, nucleic acid amplification techniques utilise enzymes (e.g. polymerases) to generate copies of a target nucleic acid that is bound specifically by one or more oligonucleotide primers. Non-limiting examples of amplification techniques in which TLM probes may be used include one or more of the polymerase chain reaction (PCR), the reverse transcription polymerase chain reaction (RT-PCR), strand displacement amplification (SDA), helicase dependent amplification (HDA), Recombinase Polymerase Amplification (RPA), loop-mediated isothermal amplification (LAMP), rolling circle amplification (RCA), transcription-mediated amplification (TMA), self-sustained sequence replication (3 SR), nucleic acid sequence based amplification (NASBA), Ligase Chain Reaction (LCR) or Ramification Amplification Method (RAM).

[0703] The skilled addressee will readily understand that the applications of TLM probes described above are provided for the purpose of non-limiting exemplification only. The TLM probes disclosed may be used in any primer-based nucleic acid amplification technique and the invention is not so limited to those embodiments specifically described.

[0704] Detection of amplicons generated using TLM probes

[0705] As discussed above, TLM probes of the present invention may be utilised in any polynucleotide amplification technique, non-limiting examples of which include the PCR, RT-PCR, SDA, HDA, RPA, LAMP, RCA, TMA, RAM, LCR, 3 SR, or NASBA.

[0706] Amplicons generated by these techniques may be detected utilising TLM probes which may be cleaved or degraded using any suitable method known in the art. Nonlimiting examples include the use of catalytic nucleic acids, exonucleases, endonucleases and the like.

[0707] A PlexZyme may be utilised to generate cleaved or modified TLM probes by detecting amplicons generated through methods such as PCR, RT-PCR, SDA, HDA, RPA, TMA, LAMP, RCA, LCR, RAM, 3 SR, and NASBA. The PlexZyme may comprise one or more partzyme(s). PlexZymes are multi-component nucleic acid enzymes which are assembled and are only catalytically active in the presence of an assembly facilitator which may be, for example, a target to be detected such as an amplicon generated from a polynucleotide sequence using primers. PlexZymes are composed of multiple partenzymes, or partzymes, which self-assemble in the presence of one or more assembly facilitators and form active PlexZymes which catalytically modify substrates. The substrate and assembly facilitators (target) are separate nucleic acid molecules. The partzymes have multiple domains including (i) sensor arms which bind to the assembly facilitator (such as a target nucleic acid); (ii) substrate arms which bind the substrate, and (iii) partial catalytic core sequences which, upon assembly, combine to provide a complete catalytic core. PlexZymes can be designed to recognise a broad range of assembly facilitators including, for example, different target nucleic acid sequences. In response to the presence of the assembly facilitator, PlexZymes modify their substrates. This substrate modification can be linked to signal generation and thus PlexZymes can generate an enzymatically amplified output signal. The assembly facilitator may be a target nucleic acid present in a biological or environmental sample (e.g. an amplicon generated from a polynucleotide target using primers). In such cases, the detection of the modification of the substrate by the PlexZyme activity is indicative of the presence of the target. Several PlexZymes capable of cleaving nucleic acid substrates are known in the art. PlexZymes and modified forms thereof are known in the art and disclosed in PCT patent publication numbers WO / 2007 / 041774, WO / 2008 / 040095, W02008 / 122084, and related US patent publication numbers 2007-0231810, 2010-0136536, and 2011-0143338 (the contents of each of these documents are incorporated herein by reference in their entirety).

[0708] Use of TLM probes as internal calibrator for machine-to-machine variation or well-to-well variation

[0709] A TLM probe could be used as an internal calibrator, since fluorescent signal could be generated by heating the reaction to above the effective Tmcleavedand below effective Tmintact of the probe. The signal could be quenched by either lowering the reaction temperature below the effective Tmcleavedor increasing the reaction temperature above effective Tmintact, independent of the presence of the target in the reaction. Therefore, if the measurements would be taken prior to target amplification where the TLM probe would be intact, the difference between the measured values could function as passive reference signal, which could be used for signal normalisation to account for the well-to-well variations.

[0710] A calibrator method that uses an TLM probe would have several advantages over other approaches including that it would not require the use of additional reagents to be added to the reaction nor would it require the use of data obtained from other wells. This method would function to calibrate and correct for well-to-well variations that may be present. Furthermore, the calibration would be processed using the data acquired in the same channel and therefore would not be affected by any channel-to-channel variations that may be present within the instrument. Where multiple channels are utilised for a multiplex reaction, each channel could be independently calibrated against the TLM probe signal in each channel. This would be favorable to a scenario where the signals are calibrated against signals in a different channel, such as conventional passive reference dye or signals from the internal control or endogenous control, as the calibration is adversely affected if the ratio of the expected signal intensity between the channels differs significantly between the instruments, causing channel-to-channel variations.

[0711] Diagnostic applications Methods using TLM Probes, optionally in combination with M-Tec and LOCS oligonucleotides and / or other well-known report probes, may be used for diagnostic and / or prognostic purposes in accordance with the methods described herein. The diagnostic and / or prognostic methods may be performed ex vivo or in vitro. However, the methods of the present invention need not necessarily be used for diagnostic and / or prognostic purposes, and hence applications that are not diagnostic or prognostic are also contemplated.

[0712] In some embodiments, the methods described herein may be used to diagnose infection in a subject. For example, the methods may be used to diagnose infection by bacteria, viruses, fungi / yeast, protists and / or nematodes in the subject. In one embodiment, the virus may be an enterovirus. The subject may be a bovine, equine, ovine, primate, avian or rodent species. For example, the subject may be a mammal, such as a human, dog, cat, horse, sheep, goat, or cow. The subject may be afflicted with a disease arising from the infection. For example, the subject may have meningitis arising from an enterovirus infection. Accordingly, methods of the present invention may in certain embodiments be used to diagnose meningitis.

[0713] The methods of the present invention may be performed on a sample. The sample may be derived from any source. For example, the sample may be obtained from an environmental source, an industrial source, or by chemical synthesis.

[0714] It will be understood that a “sample” as contemplated herein includes a sample that is modified from its original state, for example, by purification, dilution or the addition of any other component or components.

[0715] The methods of the present invention including, but not limited to diagnostic and / or prognostic methods, may be performed on a biological sample. The biological sample may be taken from a subject. Stored biological samples may also be used. Non-limiting examples of suitable biological samples include whole blood or a component thereof (e.g. blood cells, plasma, serum), urine, cervico-vaginal mucus, stool, saliva, lymph, bile fluid, sputum, tears, cerebrospinal fluid, bronchioalveolar lavage fluid, synovial fluid, semen, ascitic tumour fluid, breast milk and pus.

[0716] Kits

[0717] The present invention provides kits comprising one or more agents for performing methods of the present invention. Typically, kits for carrying out the methods of the present invention contain all the necessary reagents to carry out the method.

[0718] In some embodiments the kits may comprise oligonucleotide components capable of forming one or more PlexZymes in the presence of an appropriate assembly facilitator(s) (e.g. an amplicon as described herein) and / or one or more TLM probes. For example, the kit may comprise a first container comprising at least a first and a second oligonucleotide component of an TLM probe and a second container comprising a third and a fourth oligonucleotide component comprising partzymes, wherein self-assembly of the third and a fourth partzymes, into a PlexZyme requires association of a first assembly facilitator (e.g. an amplicon) present in a test sample. Optionally, for example, the kit may comprise at least a fifth and sixth oligonucleotide component comprising a third and fourth partzyme, and a second container comprising a M-Tec substrate, wherein self-assembly of the third and fourth partzymes, and the M-Tec substrate, into an PlexZyme M-Tec complex requires association of a second assembly facilitator (e.g. an amplicon) present in a test sample. Optionally, for another example, the kit may comprise at least a seventh and eighth oligonucleotide component comprising a third and fourth partzyme, and a second container comprising a LOCS substrate, wherein self-assembly of the third and fourth partzymes, and the LOCS substrate, into an PlexZyme LOCS complex requires association of a third assembly facilitator (e.g. an amplicon) present in a test sample. Accordingly, in such an embodiment, the first and second partzymes, and an oligonucleotide component for a substrate within the Loop region, may be applied to the test sample in order to determine the presence of one or more target amplicons. In general, the kits comprise at least a first and second oligonucleotide component for a TLM probe, provided herein. In some examples, the kit comprises a first oligonucleotide component and a second oligonucleotide component of a TLM probe.

[0719] The kit may comprise a first oligonucleotide component, a second oligonucleotide component and a third oligonucleotide component of a TLM probe. In some examples, the kit comprises a first container comprising a first component oligonucleotide, a second component oligonucleotide and optionally a third oligonucleotide of a TLM probe (e.g., a TLM-P probe), and a second container comprising partzyme oligonucleotides capable of self-assembly into an MNAzyme in the presence of a first assembly facilitator (e.g., an amplicon). In some examples, the kit comprises a first container comprising a first oligonucleotide component, a second oligonucleotide component and optionally a third oligonucleotide component of an TLM probe (e.g., a TLM-H probe), and a second container comprising an exonuclease or a polymerase having exonuclease activity. In some examples, the kit comprises a first container comprising a first oligonucleotide component, a second oligonucleotide component and optionally a third oligonucleotide component of a TLM probe (e.g., a TLM-E probe), and a second container comprising an endonuclease, such as a restriction enzyme or a nicking endonuclease. Typically, the kits of the present invention will also comprise other reagents, wash reagents, enzymes and / or other reagents as required in the performance of the methods of the invention such as PCR or other nucleic acid amplification techniques.

[0720] The kits may be fragmented kits or combined kits as defined herein.

[0721] Fragmented kits comprise reagents that are housed in separate containers, and may include small glass containers, plastic containers or strips of plastic or paper. Such containers may allow the efficient transfer of reagents from one compartment to another compartment whilst avoiding cross-contamination of the samples and reagents, and the addition of agents or solutions of each container from one compartment to another in a quantitative fashion.

[0722] Such kits may also include a container which will accept the test sample, a container which contains the reagents used in the assay, containers which contain wash reagents, and containers which contain a detection reagent.

[0723] Combined kits comprise all of the components of a reaction assay in a single container (e.g. in a single box housing each of the desired components).

[0724] A kit of the present invention may also include instructions for using the kit components to conduct the appropriate methods. Kits and methods of the invention may be used in conjunction with automated analysis equipment and systems, for example, including but not limited to, real time PCR machines.

[0725] For application to amplification, detection, identification or quantitation of different targets, a single kit of the invention may be applicable, or alternatively different kits, for example containing reagents specific for each target, may be required. Methods and kits of the present invention find application in any circumstance in which it is desirable to detect, identify or quantitate any entity.

[0726] It will be appreciated by persons of ordinary skill in the art that numerous variations and / or modifications can be made to the present invention as disclosed in the specific embodiments without departing from the spirit or scope of the present invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

[0727] Examples

[0728] The present invention will now be further described in greater detail by reference to the following specific examples, which should not be construed as in any way limiting the scope of the invention. Example 1: Detection of a target using an TLM probe which generates signal at one temperature only.

[0729] The following example demonstrates how a TLM probe can be used to detect the presence of a target (Chlamydia trachomatis,' CT) by monitoring increases in the fluorescence signal at second ‘middle’ temperature (61 °C) only in the presence of target, while there’s no change in signal at the first Tow’ (45°C) or third ‘high’ (78°C) temperature regardless of presence or absence of target.

[0730] Oligonucleotides

[0731] The oligonucleotides for amplification and detection of Target 1 (CT) specific to this experiment include: Forward Primer 1 (SEQ ID: 1) Reverse Primer 1 (SEQ ID: 2), Partzyme Al (SEQ ID: 3), Partzyme Bl (SEQ ID: 4), OC1 / 1-Q1 (SEQ ID: 5), OC2 / 1-FAM (SEQ ID: 6). The sequences are listed in Table 7.

[0732] Reaction conditions

[0733] Real-time detection of the target sequence was performed in a total reaction volume of 20 pL using a BioRad® CFX96 thermocycler. The cycling parameters were 95°C for 30 seconds, 50 cycles of 95°C for 5 seconds, 45°C for 40 seconds, 61°C for 10 seconds, and 78°C for 3 sec. Fluorescence data was acquired in the FAM Channel at the 45°C, 61 °C and 76°C step at each PCR cycle. All reactions were run in duplicates and contained 40 nM of Forward Primer 1, 200 nM of each of Reverse Primer 1, Partzyme Al, Partzyme Bl, OC1 / 1-Q1 and OC2 / 1-FAM, lx NH4 buffer (Meridian Bioscience), 8 mM MgCh (Sigma-Aldrich), 800 pM dNTP mix (Meridian Bioscience) and 2 U AptaTaq exo- DNA polymerase (Roche CustomBiotech).

[0734] The reactions either contained no target (NF H2O), or 10000 or 40 copies of synthetic double-stranded DNA fragments (IDT), which is homologous to the target gene (CT). All reactions except for the no target control (NF H2O) contained a background of 10000 copies of human genomic DNA (Promega).

[0735] Results

[0736] During PCR amplification, fluorescence was measured at three temperatures in realtime. The presence of CT target was detected and monitored by the increase in fluorescence acquired at 61°C. No change in fluorescence signal was observed at 45°C or 76°C, regardless of the presence or absence of the target. The signal observed at 45°C was generated by cleavage of substrate sequence within the Oligo 1 by PlexZymes assembled in the presence of target CT template. Cleavage resulted in separation of the fluorophore and quencher moieties in the presence, but not in the absence, of target as illustrated schematically in Figure 5 (ii) and (v) respectively. The first Tow’ temperature of 45°C is below the effective Tmcleavedand hence Oligo 2 remains hybridised to either a cleaved fragment of Oligo 1 when target is present, or an intact uncleaved Oligo 1 in the absence of target as illustrated schematically in Figure 5 (i) and (iv) respectively. No change in signal was observed at 78°C because all reactions give the same baseline fluorescence regardless of whether target is present or absent as illustrated schematically in Figure 5 (iii) and (vi) respectively. Background fluorescence at the high temperature is the result of separation of Oligo 1 and Oligo 2 because 78°C is above the effective Tmintact.

[0737] Experimental results in Figure 12 shows amplification curves where fluorescence was acquir...

Claims

CLAIMS1. A method for determining the presence or absence of a target in a sample, the method comprising:(a) preparing a mixture for a reaction by contacting the sample or a derivative thereof putatively comprising the target with:- a temperature-dependent looped multi-component probe (TLM) comprising a first oligonucleotide component and a second oligonucleotide component,wherein the first oligonucleotide component comprises a first capture region and a second capture region, the first capture region being capable of hybridisation to a first capture region of the second oligonucleotide component by complementary base pairing to form a first double-stranded portion, and the second capture region being capable of hybridisation to a third capture region of the first oligonucleotide component or to a second capture region of the second oligonucleotide component by complementary base pairing to form a second double-stranded portion,wherein the first oligonucleotide component further comprises a single-stranded loop portion of unhybridised nucleotides comprising a sensor region capable of serving as a substrate for an enzyme, wherein the sensor region is located between the first and second capture regions of the first oligonucleotide component;wherein the first oligonucleotide component is connected to a first detection moiety and the second oligonucleotide component is connected to a second detection moiety; wherein the first detection moiety of the first oligonucleotide is connected to the second capture region of the first oligonucleotide;- an enzyme capable of modifying the sensor region of the first oligonucleotide component only when the target is present in the sample;(b) treating the mixture under conditions suitable for the enzyme to modify the sensor region of the first oligonucleotide component to thereby generate a first fragment comprising the first capture region of the first oligonucleotide and a second fragment connected to the first detection moiety, thereby enabling the first and second detection moieties to spatially separate and generate a first detectable signal,(c) measuring a level of background signal or detectable signal generated at a defined temperature at or below which the first capture region is hybridised to the second oligonucleotide component; and(d) determining the presence or absence of the target based upon the level of detectable signal measured at the defined temperature, wherein a detectable signal at the defined temperature is indicative of the presence of the target in the sample.

2. The method of claim 1 wherein the enzyme is capable of digesting the sensor region of the first oligonucleotide component only when the target is present in the sample, and wherein step (b) comprises treating the mixture under conditions suitable for the enzyme to digest the sensor region of the first oligonucleotide component to thereby generate a first fragment comprising the first capture region of the first oligonucleotide and a second fragment connected to the first detection moiety.

3. The method of claim 1 or claim 2 wherein the method comprises:(i) measuring a level of background signal or detectable signal generated by the first and second detection moieties in the mixture at the defined temperature- at a timepoint prior to or during said treating the mixture, and- at one or more subsequent timepoint(s) during or following said treating the mixture; and (ii) determining a presence of or a change in the level of detectable signal which differs from the background signal and is indicative of the presence of the target in the sample.

4. The method of claim 3 wherein step (c) comprises measuring the detectable signal or any said background signal:- at one or more timepoints prior to said treating;- at one or more timepoints during said treating;- at one or more timepoints after said treating;- at one or more timepoints during said treating and at one or more timepoints after said treating;- at one or more timepoints prior to said treating and at one or more timepoints after said treating; or- at one or more timepoints before and during said treating and at one or more timepoints after said treating.

5. The method of claim 3 or claim 4 wherein step (d) comprises using a predetermined threshold value to determine if the detectable signal differs from any said background signal at the defined temperature.

6. The method of claim 1 or claim 2 further comprising measuring a level of control background signal generated at the defined temperature in a control mix;wherein step (c) comprises measuring a level of the background or detectable signal in the mixture contacted by the sample or derivative thereof; andwherein step (d) comprises determining whether a detectable signal that differs from the control background signal is generated and indicative of the presence of the target in the sample.

7. The method of claim 1 or claim 2 further comprising:measuring a level of control background signal generated at the defined temperature in a control mix, anddetermining whether the level of control background signal measured in the control mix differs from the level of background signal or detectable signal measured in the mixture at step (c),wherein a difference in the level of background signal or detectable signal measured in the mixture at step (c) compared to the level of control background signal measured in the control mix is indicative of the presence of the target in the sample.

8. The method of claim 6 or claim 7 wherein the control mix does not comprise the target but is otherwise equivalent to the mixture.

9. The method of claim 6 or claim 7 wherein the control mix does not comprise the enzyme but is otherwise equivalent to the mixture.

10. The method of claim 1 or claim 2 further comprising:measuring a level of control detectable signal generated at the defined temperature in a control mix, wherein the control mix comprises a predetermined amount of the target but is otherwise equivalent to the mixture; anddetermining whether the level of control detectable signal measured in the control mix differs from the level of background signal or detectable signal measured in the mixture at step (c),wherein a difference in the level of background signal or detectable signal measured in the mixture at step (c) compared to the level of control detectable signal measured in the control mix is indicative of the presence and / or amount of the target in the sample.

11. The method of any one of claims 1 to 10 wherein the target is a nucleic acid and at least a portion of the sensor region hybridises to a complementary sequence in the target to thereby form a duplex between the sensor region and the target.

12. The method of claim 11 wherein the enzyme is an endonuclease that recognises a sequence in the duplex.

13. The method of claim 12 wherein the endonuclease digests at least one strand of the duplex to thereby form the first and second fragments.

14. The method of claim 12 wherein the endonuclease is a nicking endonuclease that digests the sensor region of the first oligonucleotide component after formation of the duplex to thereby form the first and second fragments.

15. The method of claim 11 wherein the enzyme is an exonuclease that hydrolyses the sensor region of the first oligonucleotide component after formation of the duplex to thereby form the first and second fragments.

16. The method of claim 15 wherein the exonuclease is a polymerase with exonuclease activity.

17. The method of claim 16 wherein- at least a portion of the sensor region hybridises to a complementary sequence in the target to thereby form a duplex between the sensor region and the target,- said mixture further comprises a target primer capable of binding to the target at a region upstream of said complementary sequence,- said treating the mixture comprises:hybridisation of the target primer to the target by complementary base pairing, extending the primer using the polymerase with exonuclease activity and using the nucleic acid target as a template,wherein the polymerase comprising exonuclease activity digests the sensor region of the first oligonucleotide component after formation of the duplex to thereby form the first and second fragments.

18. The method of any one of claims 1 to 10 wherein the enzyme is a DNAzyme.

19. The method of any one of claims 1 to 10 wherein the target is a nucleic acid and the sensor region of the first oligonucleotide component is not complementary to the target.

20. The method of any one of claims 1 to 10 or 19 wherein the target is a nucleic acid and the enzyme is a multi-component nucleic acid enzyme (MNAzyme) comprising two partzyme oligonucleotides capable of self-assembling to form the MNAzyme only in the presence of the target.

21. The method of claim 20 wherein said treating comprises:hybridising sensor arms of the MNAzyme to the target by complementary base pairing, and hybridising substrate arms of the MNAzyme to at least a portion of the sensor region of the first oligonucleotide component by complementary base pairing to facilitate cleavage of the first oligonucleotide component and generation of the first and second fragments.

22. The method of any one of claims 1 to 10 wherein the target is a nucleic acid.

23. The method of claim 22 wherein the target is an amplicon of a nucleic acid.

24. The method of claim 23 wherein the amplicon is produced by an amplification reaction selected from the group consisting of polymerase chain reaction (PCR), strand displacement amplification (SDA), nicking enzyme amplification reaction (NEAR), helicase dependent amplification (HD A), Recombinase Polymerase Amplification (RPA), loop-mediated isothermal amplification (LAMP), rolling circle amplification (RCA), transcription-mediated amplification (TMA), self-sustained sequence replication (3 SR), nucleic acid sequence based amplification (NASBA), Ligase Chain Reaction (LCR) or Ramification Amplification Method (RAM) and reverse transcription polymerase chain reaction (RT-PCR).

25. The method of claim 24, wherein said detecting:- occurs prior to said amplification or within 1, 2, 3, 4, or 5 cycles of said amplification commencing; and / or- occurs after completion of said amplification.

26. The method of any one of claims 23 to 25 wherein said determining the presence or absence of the target comprises a melt curve analysis.

27. The method of any one of claims 1 to 10 wherein:- the enzyme is a DNAzyme or a ribozyme requiring a co-factor for catalytic activity, - said treating of the mixture comprises using conditions suitable for:binding of the cofactor to the DNAzyme or ribozyme to render it catalytically active, hybridisation of the DNAzyme or ribozyme to the first oligonucleotide component by complementary base pairing,catalytic activity of the DNAzyme or ribozyme to thereby digest the first oligonucleotide component and generate the first fragment and the second fragment, and- the target is the co-factor.

28. The method of claim 27 wherein the co-factor is a metal ion, such as a metal ion selected from: Mg2+, Mn2+, Ca2+ and Pb2+.

29. The method of any one of claims 1 to 10 wherein the enzyme is an aptazyme wherein:- the sensor region comprises a substrate for an aptazyme;- the target is an analyte, protein, peptide, compound or nucleic acid;- the mixture comprises an aptazyme comprising an aptamer capable of binding to the target; and- said treating the mixture further comprises binding of the aptazyme to the target and to the sensor region to facilitate cleavage of the first oligonucleotide component to thereby generate the first fragment and the second fragment.

30. A method for determining the presence or absence of a target in a sample, the method comprising:(a) preparing a mixture for a reaction by contacting the sample or a derivative thereof putatively comprising the target with:- a temperature-dependent looped multi-component probe (TLM) comprising a first oligonucleotide component and a second oligonucleotide component,wherein the first oligonucleotide component comprises a first capture region and a second capture region, the first capture region being capable of hybridisation to a first capture region of the second oligonucleotide component by complementary base pairing to form a first double-stranded portion, and the second capture region being capable of hybridisation to a third capture region of the first oligonucleotide component or to a second capture region of the second oligonucleotide component by complementary base pairing to form a second double-stranded portion,wherein the first oligonucleotide component further comprises a single-stranded loop portion of unhybridised nucleotides comprising a sensor region capable of serving as a substrate for an enzyme, wherein the sensor region is located between the first and second capture regions of the first oligonucleotide component,wherein the first oligonucleotide component is connected to a first detection moiety and the second oligonucleotide component is connected to a second detection moiety; wherein the first detection moiety of the first oligonucleotide is connected to the second capture region of the first oligonucleotide;- an enzyme capable of modifying the sensor region of the first oligonucleotide component only when the target is present in the sample;(b) treating the mixture under conditions suitable for the enzyme to modify the sensor region of the first oligonucleotide component to thereby generate a first fragment comprising the first capture region of the first oligonucleotide and a second fragment connected to the first detection moiety, thereby enabling the first and second detection moieties to spatially separate and generate a first detectable signal,(c) measuring a level of detectable signal generated at a defined temperature at or below which the first capture region hybridises to the second oligonucleotide component; and(d) determining the presence or absence of the target based upon the presence or absence of a change in detectable signal generating a melt curve peak, wherein absence of a melt curve peak is indicative of the presence of the target in the sample and presence of a melt curve peak is indicative of the absence of the target in the sample.

31. The method of any one of claims 1 to 30 wherein the second oligonucleotide component is directly labelled with the second detection moiety.

32. The method of any one of claims 1 to 31 wherein the first fragment is not directly labelled with a detection moiety.

33. The method of any one of claims 1 to 32 wherein the second fragment is not directly labelled with the first detection moiety.

34. The method of any one of claims 1 to 33 wherein the first oligonucleotide component is not directly labelled with the first detection moiety.

35. The method of any one of claims 1 to 34 wherein the second capture region of the first oligonucleotide component is capable of hybridisation to a third capture region of the first oligonucleotide component by complementary base pairing to form the second doublestranded portion.

36. The method of any one of claims 1 to 34 wherein the first oligonucleotide further comprises a third capture region capable of hybridisation to the second capture region of the first oligonucleotide component by complementary base pairing to form the second double-stranded portion.

37. The method of any one of claims 1 to 34 wherein the second capture region of the first oligonucleotide component is capable of hybridisation to a second capture region of the second oligonucleotide component by complementary base pairing to form the second double-stranded portion.

38. The method of any one of claims 1 to 37 wherein the first double-stranded portion of the TLM probe has a melting temperature (Tm) that is above the defined temperature.

39. The method of any one of claims 1 to 38 wherein the Tm of the second double-stranded portion is less than the Tm of the first double-stranded portion.

40. The method of any one of claims 1 to 33 wherein the first oligonucleotide component is directly labelled with the first detection moiety.

41. The method of any one of claims 1 to 40 wherein the TLM probe does not comprise more than two detection moieties.

42. The method of any one of claims 1 to 41 wherein the first oligonucleotide component is not directly labelled with more than one detection moiety43. The method of any one of claims 1 to 41 wherein the first oligonucleotide component is not connected to more than one detection moiety.

44. The method of any one of claims 1 to 43 wherein the second oligonucleotide component is not directly labelled with more than one detection moiety or is not connected to more than one detection moiety.

45. The method of any one of claims 1 to 44 wherein:the first detection moiety is a fluorophore, and the second detection moiety is a quencher; orthe first detection moiety is a quencher, and the second detection moiety is a fluorophore,optionally wherein the TLM probe does not comprise more than one quencher or optionally wherein the detectable signal is fluorescence emitted in the presence of the target.

46. The method of any one of claims 1 to 45 wherein neither the first oligonucleotide component or the second oligonucleotide component serve as a primer for a DNA polymerase in an extension reaction and / or wherein neither the first oligonucleotide component or the second oligonucleotide serve as a template for a DNA polymerase in an extension reaction.

47. The method of any one of claims 1 to 46 wherein the presence or absence of the target in a sample is determined at temperatures above the melting temperature (Tm) of the cleaved TLM probe and below the Tm of the intact TLM probe.

48. The method of any one of claims 1 to 47 wherein the second oligonucleotide component is not enzymatically cleaved or degraded.

49. The method of any one of claims 1 to 48 wherein the sensor region is located between the first capture region of the first oligonucleotide component and the first detection moiety or between the second capture region of the first oligonucleotide component and the third capture region of the first oligonucleotide component.

50. The method of any one of claims 1 to 49 wherein following said treating the mixture the first fragment is capable of hybridising to the second oligonucleotide component via the first capture region.

51. The method of any one of claims 1 to 50 wherein the second capture region of the first oligonucleotide is capable of hybridisation to a third capture region of the first oligonucleotide component by complementary base pairing to form a second doublestranded portion, and the probe further comprises a third oligonucleotide component comprising a first capture region capable of hybridisation to a fourth capture region of the first oligonucleotide component by complementary base pairing to form a third doublestranded portion.

52. The method of any one of claims 1 to 50 wherein the second capture region of the first oligonucleotide is capable of hybridisation to a second capture region of the second oligonucleotide component by complementary base pairing to form a second doublestranded portion, and the probe further comprises a third oligonucleotide componentcomprising a first capture region capable of hybridisation to a third capture region of the first oligonucleotide component by complementary base pairing to form a third doublestranded portion.

53. The method of claim 51 or 52 wherein the third oligonucleotide is connected to a detection moiety or wherein the third oligonucleotide is directly labelled with a detection moiety.

54. The method of claim 53 wherein the detection moiety is the first or the second detection moiety, optionally wherein the first detection moiety is a fluorophore, and the second detection moiety is a quencher; or the first detection moiety is a quencher, and the second detection moiety is a fluorophore.

55. The method of any one of claims 1 to 54 wherein the biological sample is obtained from a subject and / or generation of the detectable signal at the defined temperature is not reversible.

56. The method of any one of claims 1 to 55 wherein the method is performed in vitro or ex vivo.

57. A method for determining the presence or absence of a first target and a second target in a sample, the method comprising:(a) preparing a mixture for a reaction by contacting the sample or a derivative thereof putatively comprising the first and / or second target with:- a temperature-dependent looped multi-component (TLM) probe for detection of the first target, the TLM probe comprising a first oligonucleotide component and a second oligonucleotide component;wherein the first oligonucleotide component comprises a first capture region and a second capture region, the first capture region being capable of hybridisation to a first capture region of the second oligonucleotide component by complementary base pairing to form a first double-stranded portion, and the second capture region being capable of hybridisation to a third capture region of the first oligonucleotide component or to a second capture region of the second oligonucleotide component by complementary base pairing to form a second double-stranded portion;wherein the first oligonucleotide component further comprises a single-stranded loop portion of unhybridised nucleotides comprising a sensor region capable of serving as a substrate for an enzyme, wherein the sensor region is located between the first and second capture regions of the first oligonucleotide component;wherein the first oligonucleotide component is connected to a first detection moiety and the second oligonucleotide component is connected to a second detection moiety;wherein the first detection moiety of the first oligonucleotide is connected to the second capture region of the first oligonucleotide;- a second nucleic acid probe for detection of the second target, the second nucleic acid probe comprising third and fourth detection moieties,wherein the first and second detection moieties are capable of generating a first detectable signal, and the third and fourth detection moieties are capable of generating a second detectable signal, and- a first enzyme capable of modifying the sensor region of the first oligonucleotide component only when the first target is present in the sample;(b) treating the mixture under conditions suitable for:- the first enzyme to modify the sensor region of the first oligonucleotide component to thereby generate a first fragment comprising the first capture region of the first oligonucleotide and a second fragment connected to the first detection moiety, thereby enabling the first and second detection moieties to spatially separate and generate a first detectable signal,- the second target to induce a modification of the second nucleic acid probe, thereby enabling the third and fourth detection moieties to spatially separate and generate a second detectable signal;(c) measuring a level of background or detectable signal:- at a first temperature at or below which the first capture region is hybridised to the second oligonucleotide component,- at a second temperature at or above which the first capture region is not hybridised to the second oligonucleotide component,(d) determining whether at one or more timepoints during or after said treating: - a first detectable signal is generated at the first temperature at or below which the first capture region is hybridised to the second oligonucleotide component,- a second detectable signal arising from said modification of the second nucleic acid probe is generated at the second temperature, wherein the second detectable signal is indicative of the presence of the second target in the sample.

58. The method of claim 57 wherein the first enzyme is capable of digesting the sensor region of the first oligonucleotide component only when the first target is present in the sample, and wherein step (b) comprises treating the mixture under conditions suitable for the first enzyme to digest the sensor region of the first oligonucleotide component to thereby generate a first fragment comprising the first capture region of the first oligonucleotide and a second fragment connected to the first detection moiety.

59. The method of claim 57 or claim 58 wherein a first detectable signal at the first temperature is indicative of the presence of the first target in the sample.

60. The method of claim 59 wherein the presence of the first target is determined at the first temperature based upon the first detectable signal generated at the first temperature.

61. The method of any one of claims 57 to 60 wherein the presence of the second target is determined at the second temperature based upon the second detectable signal generated at the second temperature.

62. The method of claim 57 or claim 58 wherein:(i) at the first temperature- a first detectable signal is generated in the presence of the first target,- a second detectable signal is generated in the presence of the second target, or- a first detectable signal and a second detectable signal is generated in the presence of both the first target and the second target; and(ii) a second detectable signal is generated at the second temperature only in the presence of the second target.

63. The method of any one of claims 57 to 62 wherein the method comprises:- measuring a level of background signal or detectable signal at the first and second temperatures generated by the first and second detection moieties and by the third and fourth detection moieties in the mixture,- determining a presence of or a change in the level of the first detectable signal which differs from the background signal and is indicative of the presence of the first target in the sample, and- determining a presence of or a change in the level of the second detectable signal arising from said modification generated at the second temperature which differs from the background signal and is indicative of the presence of the second target in the sample.

64. The method of any one of claims 57 to 63 wherein at the second temperature, dissociation of the second oligonucleotide component from the capture region of either the first oligonucleotide component present in the absence of the first target, or the first fragment generated by modification of the first oligonucleotide component in the presence of the first target generate an equal, similar or equivalent background signal.

65. The method of any one of claims 57 to 64 wherein step (c) comprises measuring a level of background signal at a third temperature and at the third temperature the second capture region remains hybridised in the presence or absence of the target, thereby generating a background signal.

66. The method of any one of claims 57 to 65 wherein said determining comprises detection of the first detectable signal and / or any said background signal:- at one or more timepoints prior to said treating;- at one or more timepoints during said treating;- at one or more timepoints after said treating;- at one or more timepoints during said treating and at one or more timepoints after said treating;- at one or more timepoints prior to said treating and at one or more timepoints after said treating; or- at one or more timepoints before and during said treating and at one or more timepoints after said treating.

67. The method of any one of claims 57 to 66, wherein said determining in part (d) comprises:- using a predetermined threshold value to determine if the first detectable signal differs from any said background signal at the first temperature; and / or- using a predetermined threshold value to determine if the second detectable signal differs from any said background signal at the second temperature.

68. The method of any one of claims 57 to 63 comprising:measuring a level of first control background signal at the first temperature provided by the first and second detection moieties and by the third and fourth detection moieties in a control mix;measuring a level of second control background signal at the second temperature provided by the first and second detection moieties and by the third and fourth detection moieties in the control mix;determining whether a level of the first detectable signal generated at the first temperature at step (c) in the mixture contacted by the sample or derivative thereof differs from the level of first control background signal measured in the control mix, wherein a difference in the level of detectable signal measured in the mixture at the first temperature at step (c) compared to the first control background signal measured in the control mix is indicative of the first target in the sample; anddetermining whether a level of the second detectable signal generated at the second temperature at step (c) in the mixture contacted by the sample or derivative thereof differs from the level of second control background signal measured in the control mix, wherein a difference in the level of detectable signal measured in the mixture at the secondtemperature at step (c) compared to the second control background signal measured in the control mix is indicative of the second target in the sample.

69. The method of claim 68 wherein the control mix does not comprise:- the first target;- the second target; or- the first and second targets,but is otherwise equivalent to the mixture.

70. The method of claim 68 wherein the control mix does not comprise the first enzyme but is otherwise equivalent to the mixture.

71. The method of any one of claims 57 to 63 further comprising:measuring a level of first detectable signal generated at the first temperature in a control mix, wherein the control mix comprises a predetermined amount ofthe first target,the second target, orthe first and second targets,but is otherwise equivalent to the mixture;measuring a level of second detectable signal generated at the second temperature in the control mix;determining whether a level of the first detectable signal generated at the first temperature at step (c) in the mixture contacted by the sample or derivative thereof differs from the level of first control detectable signal measured in the control mix, wherein a difference in the level of detectable signal measured in the mixture at the first temperature at step (c) compared to the first control detectable signal measured in the control mix is indicative of the first target in the sample; anddetermining whether a level of the second detectable signal generated at the second temperature at step (c) in the mixture contacted by the sample or derivative thereof differs from the level of second control detectable signal measured in the control mix, wherein a difference in the level of detectable signal measured in the mixture at the second temperature at step (c) compared to the second control detectable signal measured in the control mix is indicative of the second target in the sample.

72. The method of any one of claims 57 to 67 wherein part (c) comprises measuring a first background signal at or within 1°C, 2°C, 3 °C, 4°C or 5°C of the first temperature, and a second background signal at or within 1°C, 2°C, 3°C, 4°C or 5°C of the second temperature.

73. The method of claim 72 wherein part (d) comprises determining whether at one or more time points during or after said treating:a first detectable signal is generated at the first temperature which differs from the first background signal and is indicative of the presence of the first target in the sample; anda second detectable signal is generated at the second temperature which differs from the second background signal and is indicative of the presence of the second target in the sample.

74. The method of any one of claims 57 to 73 wherein at the first temperature the third and fourth detection moi eties do not generate a signal which differs from the background signal.

75. The method of any one of claims 57 to 74 wherein at the second temperature the first and second detection moieties do not generate a signal which differs from the background signal.

76. The method of any one of claims 57 to 75 wherein the first and second detectable signals are detectable by a single detector optionally wherein the first and second detectable signals are detectable in the same fluorescent channel.

77. The method of any one of claims 57 to 76 wherein the first and second detectable signals are detectable as fluorescent emission at a single wavelength.

78. The method of any one of claims 57 to 77, wherein the first and second detection moieties, and the third and fourth detection moieties emit a detectable signal at the same or similar wavelength which can be detected in the same fluorescence channel.

79. The method of any one of claims 57 to 78 wherein the second oligonucleotide component is directly labelled with the second detection moiety.

80. The method of any one of claims 57 to 79 wherein the first fragment is not directly labelled with a detection moiety.

81. The method of any one of claims 57 to 79 wherein the second fragment is not directly labelled with the first detection moiety; or wherein the first oligonucleotide component is not directly labelled with the first detection moiety.

82. The method of any one of claims 57 to 81 wherein the second capture region of the first oligonucleotide component is capable of hybridisation to a third capture region of the first oligonucleotide component by complementary base pairing to form the second doublestranded portion.

83. The method of any one of claims 57 to 81 wherein the first oligonucleotide further comprises a third capture region capable of hybridisation to the second capture region of the first oligonucleotide component by complementary base pairing to form the second double-stranded portion.

84. The method of any one of claims 57 to 81 wherein the second capture region of the first oligonucleotide component is capable of hybridisation to a second capture region of the second oligonucleotide component by complementary base pairing to form the second double-stranded portion.

85. The method of claim any one of claims 82 to 84 wherein the first double-stranded portion of the TLM probe has a Tm that is above the first temperature.

86. The method of any one of claims 82 to 84 wherein the first double-stranded portion and / or the second double-stranded portion of the TLM probe have a Tm that is below the second temperature.

87. The method of any one of claims 82 to 86 wherein the Tm of the first double-stranded portion is higher than the Tm of the second double-stranded portion.

88. The method of any one of claims 82 to 86 wherein the Tm of the second double-stranded portion is less than the Tm of the first double-stranded portion.

89. The method of any one of claims 57 to 80 wherein the first oligonucleotide component is directly labelled with the first detection moiety.

90. The method of any one of claims 57 to 80 wherein the TLM probe does not comprise more than two detection moieties.

91. The method of any one of claims 57 to 90 wherein the first oligonucleotide component is not directly labelled with more than one detection moiety.

92. The method of any one of claims 57 to 91 wherein the first oligonucleotide component is not connected to more than one detection moiety.

93. The method of any one of claims 57 to 92 wherein the second oligonucleotide component is not directly labelled with more than one detection moiety.

94. The method of any one of claims 57 to 93 wherein the second oligonucleotide component is not connected to more than one detection moiety.

95. The method of any one of claims 57 to 94 wherein the second nucleic acid probe is directly labelled with the third and fourth detection moieties.

96. The method of any one of claims 57 to 94 wherein the second nucleic acid probe is not directly labelled with the third and fourth detection moieties.

97. The method of any one of claims 57 to 96 wherein:the first detection moiety is a fluorophore, and the second detection moiety is a quencher; orthe first detection moiety is a quencher, and the second detection moiety is a fluorophore.

98. The method of any one of claims 57 to 97 wherein:the first detection moiety is a fluorophore, and the second detection moiety is a quencher; orthe first detection moiety is a quencher, and the second detection moiety is a fluorophore;and wherein the TLM probe does not comprise more than one quencher.

99. The method of any one of claim 57 to 98 wherein:the first detection moiety is a fluorophore, and the second detection moiety is a quencher; orthe first detection moiety is a quencher, and the second detection moiety is a fluorophore;and wherein the first detectable signal is fluorescence emitted in the presence of the first target.

100. The method of any one of claims 57 to 99 wherein neither the first oligonucleotide component, the second oligonucleotide component nor the third oligonucleotide component serve as a primer for a DNA polymerase in an extension reaction.

101. The method of any one of claims 57 to 100 wherein neither the first oligonucleotide component, the second oligonucleotide component nor the third oligonucleotide component serve as a template for a DNA polymerase in an extension reaction.

102. The method of any one of claims 57 to 101 wherein neither the second oligonucleotide component nor the third oligonucleotide component is enzymatically cleaved or degraded.

103. The method of any one of claims 57 to 102 wherein the sensor region is located between the first capture region of the first oligonucleotide and the first detection moiety or between the second capture region of the first oligonucleotide component and the third capture region of the first oligonucleotide component.

104. The method of any one of claims 57 to 103 wherein following said treating the first fragment is capable of hybridising to the second oligonucleotide component via the first capture region.

105. The method of any one of claims 57 to 103 wherein the second capture region of the first oligonucleotide is capable of hybridisation to a third capture region of the first oligonucleotide component by complementary base pairing to form a second doublestranded portion, and the probe further comprises a third oligonucleotide component comprising a first capture region capable of hybridisation to a fourth capture region of the first oligonucleotide component by complementary base pairing to form a third doublestranded portion.

106. The method of any one of claims 57 to 103 wherein the second capture region of the first oligonucleotide is capable of hybridisation to a second capture region of the second oligonucleotide component by complementary base pairing to form a second doublestranded portion, and the probe further comprises a third oligonucleotide component comprising a first capture region capable of hybridisation to a third capture region of the first oligonucleotide component by complementary base pairing to form a third doublestranded portion.

107. The method of claim 105 or 106 wherein the third oligonucleotide is connected to a detection moiety or wherein the third oligonucleotide is directly labelled with a detection moiety.

108. The method of claim 107 wherein the detection moiety is the first or the second detection moiety, optionally wherein the first detection moiety is a fluorophore, and the second detection moiety is a quencher; or the first detection moiety is a quencher, and the second detection moiety is a fluorophore.

109. The method of any one of claims 57 to 108 wherein:the third detection moiety is a fluorophore, and the fourth detection moiety is a quencher; orthe third detection moiety is a quencher, and the fourth detection moiety is a fluorophore.

110. The method of any one of claims 57 to 109 wherein the first target is a nucleic acid and at least a portion of the sensor region hybridises to a complementary sequence in the first target to thereby form a duplex between the sensor region and the first target.

111. The method of claim 110 wherein the first enzyme is an endonuclease that recognises a sequence in the duplex, preferably wherein the endonuclease digests the duplex to thereby form the first and second fragments, more preferably wherein the endonuclease is a nicking endonuclease that digests the sensor region of the first oligonucleotide component after formation of the duplex to thereby form the first and second fragments.

112. The method of claim 110 wherein the first enzyme is an exonuclease that hydrolyses the sensor region of the first oligonucleotide component after formation of the duplex to thereby form the first and second fragments, optionally wherein the exonuclease is a polymerase with exonuclease activity.

113. The method of claim 112 wherein- the first target is a nucleic acid,- at least a portion of the sensor region hybridises to a complementary sequence in the first target to thereby form a duplex between the sensor region and the first target,- said mixture further comprises a first target primer capable of binding to the first target at a region upstream of said complementary sequence,- said treating the mixture comprises:hybridisation of the first target primer to the first target by complementary base pairing, extending the primer using the polymerase with exonuclease activity and using the first target as a template,wherein the polymerase comprising exonuclease activity digests the sensor region of the first oligonucleotide component after formation of the duplex.

114. The method of any one of claims 57 to 110 wherein the first enzyme is a DNAzyme.

115. The method of any one of claims 57 to 110 wherein the first target is a nucleic acid and the sensor region of the first oligonucleotide component is not complementary to the first target or wherein the first target is a nucleic acid and the first enzyme is a first target multi-component nucleic acid enzyme (MNAzyme) comprising two partzyme oligonucleotides capable of self-assembling to form the first target MNAzyme only in the presence of the first target.

116. The method of claim 115 wherein said treating comprises:hybridising sensor arms of the first target MNAzyme to the first target by complementary base pairing, andhybridising substrate arms of the first target MNAzyme to at least a portion of the sensor region of the first oligonucleotide component by complementary base pairing to facilitate cleavage of the first oligonucleotide component and generation of the first and second fragments.

117. The method of any one of claims 57 to 116 wherein:- the second nucleic acid probe is a substrate for a second target multi-component nucleic acid enzyme (MNAzyme), the second target MNAzyme comprising two partzyme oligonucleotides capable of self-assembling to form the second target MNAzyme only in the presence of the second target;- the mixture further comprises:the second target MNAzyme capable of cleaving the second nucleic acid probe only when the second target is present in the sample;- said treating further comprises:hybridising sensor arms of the second target MNAzyme to the second target by complementary base pairing, andhybridising substrate arms of the second target MNAzyme to the second nucleic acid probe by complementary base pairing to facilitate cleavage of the second nucleic acid probethereby providing said modification to the second nucleic acid probe and enabling the third and fourth detection moieties to spatially separate and generate the second detectable signal.

118. The method of claim 117 wherein the second nucleic acid probe is a stem-loop oligonucleotide comprising a double-stranded stem portion of hybridised nucleotides, opposing strands of which are linked by an unbroken single-stranded loop portion of unhybridised nucleotides of which all or a portion of which is complementary to the substrate arms of the second target MNAzyme.

119. The method of claim 118 wherein the stem-loop oligonucleotide is an intact stem-loop oligonucleotide and said modification comprises cleavage of a sensor region in the loop portion and the formation of a split stem-loop oligonucleotide.

120. The method of any one of claims 57 to 116 wherein:- the second target is a nucleic acid,- the second nucleic acid probe is a stem-loop oligonucleotide comprising a double-stranded stem portion of hybridised nucleotides opposing strands of which are linked by an unbroken single-stranded loop portion of unhybridised nucleotides of which all or a portion is complementary to the second target,- the mixture further comprises a polymerase with exonuclease activity,- said treating the mixture comprises using conditions suitable for:hybridisation of the second target to the single-stranded loop portion of the stem-loop oligonucleotide by complementary base pairing to form a first double-stranded sequence comprising a portion of the second target,hybridisation of a primer to the second target to form a second double-stranded sequence located upstream relative to the first double-stranded sequence comprising the portion of the second target,extending the primer using the polymerase with exonuclease activity and using the second target as a template,wherein the polymerase comprising exonuclease activity digests the single-stranded loop portion of the first double-stranded sequence and thereby forms a split stem-loop oligonucleotide.

121. The method of any one of claims 57 to 116, wherein:- the second target is a nucleic acid,- the second nucleic acid probe is a stem-loop oligonucleotide comprising a double-stranded stem portion of hybridised nucleotides opposing strands of which are linked by an unbrokensingle-stranded loop portion of unhybridised nucleotides of which all or a portion is complementary to the second target,- the mixture further comprises an endonuclease, and- said treating the mixture comprises using conditions suitable for:hybridisation of the second target to the single-stranded loop portion of the stem-loop oligonucleotide by complementary base pairing to form a double-stranded sequence comprising a portion of the second target,association of the endonuclease with the double-stranded sequence comprising a portion of the second target, andcatalytic activity of endonuclease allowing it to digest the single-stranded loop portion of the double-stranded sequence and thereby form a split stem-loop oligonucleotide.

122. The method of any one of claims 118 to 121 wherein:- the stem portion of the intact stem-loop oligonucleotide has a melting temperature (Tm) that is above the Tm of the stem portion of the split stem-loop oligonucleotide;- the first temperature is below the Tm of the stem portion of the intact stem-loop oligonucleotide, and the stem portion of the split stem-loop oligonucleotide;- the second temperature is below the Tm of the stem portion of the intact stem-loop oligonucleotide, and is above the Tm of the stem portion of the split stem-loop oligonucleotide; and- the first temperature is below the second temperature.

123. The method of any one of claims 118 to 122 wherein the Tm of the stem portion of the split stem-loop oligonucleotide is above the first temperature.

124. The method of any one of claims 118 to 123 wherein the Tm of the stem portion of the intact and split stem-loop oligonucleotide(s) is above the Tm of the first double-stranded portion of the TLM probe.

125. The method of any one of claims 57 to 116 wherein:the second nucleic acid probe is a stem-loop oligonucleotide comprising a double-stranded stem portion of hybridised nucleotides opposing strands of which are linked by an unbroken single-stranded loop portion of unhybridised nucleotides of which all or a portion is complementary to the second target, and whereinthe modification of the second nucleic acid probe is a conformational change arising from hybridisation of the second target to the single-stranded loop portion by complementary base pairing that causes spatial separation of the third and fourth detection moieties.

126. The method of claim 125 wherein the conformational change is dissociation of the opposing strands in the double-stranded stem portion of the second nucleic acid probe.

127. The method of any one of claims 118 to 126 wherein the third and fourth detection moieties are connected to opposing strands of the double-stranded stem portion of the second nucleic acid probe.

128. The method of any one of claims 57 to 116, wherein:- the second target is a nucleic acid,- the mixture further comprises:a primer complementary to a first sequence in the second target, a pitcher oligonucleotide comprising a region complementary to a second sequence in the second target that differs from the first sequence, and a tag portion that is not complementary to the second target,a first polymerase comprising exonuclease activity, andoptionally a second polymerase, and- said treating the mixture comprises:conditions suitable to hybridise the primer and the pitcher oligonucleotide to the second target,extending the primer using the first or second polymerase and the second target as a template to thereby cleave off the tag portion,hybridising the cleaved tag portion to the second nucleic acid probe by complementary base pairing,and extending the tag portion using the polymerase and the second nucleic acid probe as a template to generate a double-stranded catcher sequence comprising the second nucleic acid probe thereby providing said modification to the second nucleic acid probe and enabling the third and fourth detection moieties to provide the second detectable signal.

129. The method of claim 128 wherein:- the double-stranded catcher sequence has a Tm that is above the first temperature; and - the second temperature is below the Tm of the double-stranded catcher sequence.

130. The method of claim 128 or claim 129 wherein said extending the tag portion spatially separates the third and fourth detection moieties to thereby generate the second detectable signal.

131. The method of any one of claims 57 to 116 wherein the second target is a nucleic acid and the second nucleic acid probe is a two-part probe comprising a first part oligonucleotide and a second part oligonucleotide, wherein:- the first part oligonucleotide is complementary to a first portion of the second target, - the second part oligonucleotide is complementary to a second portion of the second target,- the first and second portions of the second nucleic acid target flank one another but do not overlap,- said treating the mixture comprises:forming a duplex structure comprising:a first double-stranded portion by hybridising the first part oligonucleotide to the second target by complementary base pairing, anda second double-stranded portion by hybridising the second part oligonucleotide to the second target by complementary base pairing,thereby bringing the first and second part oligonucleotides into proximity and providing said modification to the second nucleic acid probe enabling the third and fourth detection moi eties to come into close proximity and generate the second detectable signal.

132. The method of claim 131 wherein the second detectable signal is a decrease in fluorescence.

133. The method of claim 131 wherein the second detectable signal is an increase in fluorescence.

134. The method of any one of claims 57 to 116, wherein:- the second target is a nucleic acid,- the second nucleic acid probe comprises a sequence that is complementary to the second target,- the mixture further comprises:a primer complementary to a portion of the second target, anda polymerase with exonuclease activity;- said treating the mixture comprises:hybridising the primer to the second target by complementary base pairing, hybridising the second nucleic acid probe to the second target by complementary base pairing,extending the primer using the polymerase and the second target as a template to thereby digest the second nucleic acid probe and provide said modification to the second nucleic acid probe enabling the third and fourth detection moieties to spatially separate and generate the second detectable signal.

135. The method of any one of claims 57 to 116, wherein:- the second target is a nucleic acid,- the mixture further comprises a restriction endonuclease capable of digesting a doublestranded duplex comprising the second target; and- said treating the mixture comprises:hybridising the second nucleic acid probe to the second target by complementary base pairing to thereby form the double-stranded duplex,digesting the duplex using the restriction endonuclease to thereby provide said modification to the second nucleic acid probe and enabling the third and fourth detection moieties to spatially separate and generate the second detectable signal.

136. The method of any one of claims 57 to 116 wherein:- the second nucleic acid probe is a second TLM probe comprising a first oligonucleotide component and a second oligonucleotide component,wherein the first oligonucleotide component of the second TLM probe comprises a first capture region and a second capture region, the first capture region being capable of hybridisation to a first capture region of the second oligonucleotide component of the second TLM probe by complementary base pairing to form a first double-stranded portion, and the second capture region being capable of hybridisation to a third capture region of the first oligonucleotide component of the second TLM probe or to a second capture region of the second oligonucleotide component of the second TLM probe by complementary base pairing to form a second double-stranded portion,wherein the first oligonucleotide component of the second TLM probe further comprises a single-stranded loop portion of unhybridised nucleotides comprising a sensor region capable of serving as a substrate for an enzyme only when the second target is present in the sample, wherein the sensor region is located between the first and second capture regions of the first oligonucleotide componentwherein the first oligonucleotide component of the second TLM probe is connected to the third detection moiety and the second oligonucleotide component of the second TLM probe is connected to the fourth detection moiety,wherein the third detection moiety of the first oligonucleotide component of the second TLM probe is connected to the second capture region of the first oligonucleotide component of the second TLM probe,- the mixture further comprises the second enzyme,- said treating of the mixture comprises:treating the mixture under conditions suitable for the enzyme to digest the sensor region of the second TLM probe to thereby generate a first fragment comprising the first capture region of the first oligonucleotide and a second fragment connected to the third detection moiety, thereby enabling the third and fourth detection moieties to spatially separate and generate a second detectable signal.

137. The method of claim 136 wherein the first enzyme is the same as the second enzyme.

138. The method of claim 136 or claim 137 wherein the double-stranded portions of the second TLM probe have a Tm above the Tm of the first double stranded portion and the second double stranded portion of the first TLM probe.

139. The method of any one of claims 57 to 138 wherein:- the first target is a nucleic acid;- the second target is a nucleic acid; or- the first target is a nucleic acid and the second target is a nucleic acid.

140. The method of claim 139 wherein the first target and / or the second target is an amplicon of a nucleic acid.

141. The method of claim 140 wherein the amplicon is produced by an amplification reaction selected from the group consisting of polymerase chain reaction (PCR), strand displacement amplification (SDA), nicking enzyme amplification reaction (NEAR), helicase dependent amplification (HD A), Recombinase Polymerase Amplification (RPA), loop-mediated isothermal amplification (LAMP), rolling circle amplification (RCA), transcription-mediated amplification (TMA), self-sustained sequence replication (3 SR), nucleic acid sequence based amplification (NASBA), Ligase Chain Reaction (LCR) or Ramification Amplification Method (RAM) and reverse transcription polymerase chain reaction (RT-PCR).

142. The method of claim 141 wherein said determining:- occurs prior to said amplification or within 1, 2, 3, 4, or 5 cycles of said amplification commencing; and / or- occurs after completion of said amplification.

143. The method of any one of claims 140 to 142 wherein said determining the presence or absence of the first and second targets comprises a melt curve analysis.

144. The method of any one of claims 57 to 116 or 136 to 143 wherein:- the mixture further comprises a DNAzyme or a ribozyme requiring a co-factor for catalytic activity;- said treating of the mixture comprises using conditions suitable for:binding of the co-factor to the DNAzyme or ribozyme to render it catalytically active, hybridisation of the DNAzyme or ribozyme to the second nucleic acid probe by complementary base pairing, andcatalytic activity of the DNAzyme or ribozyme to thereby digest the second nucleic acid probe and thereby provide said modification to the second nucleic acid probe enabling the third and fourth detection moieties to spatially separate and generate the second detectable signal, and- the second target is the co-factor.

145. The method of any one of claims 57 to 110 or 117 to 144 wherein:- the first enzyme is a DNAzyme or a ribozyme requiring a co-factor for catalytic activity,- said treating of the mixture comprises using conditions suitable for:binding of the cofactor to the DNAzyme or ribozyme to render it catalytically active, hybridisation of the DNAzyme or ribozyme to the first oligonucleotide component by complementary base pairing,catalytic activity of the DNAzyme or ribozyme to thereby digest the first oligonucleotide component and generate the first fragment and the second fragment, and- the first target is the co-factor.

146. The method of claim 144 or claim 145 wherein the co-factor is a metal ion, such as a metal ion selected from: Mg2+, Mn2+, Ca2+ and Pb2+.

147. The method of any one of claims 57 to 106 or 117 to 144 wherein the first enzyme is an aptazyme wherein:- the sensor region comprises a substrate for an aptazyme;- the first target is an analyte, protein, peptide, compound or nucleic acid;- the mixture comprises an aptazyme comprising an aptamer capable of binding to the first target; and- said treating the mixture further comprises binding of the aptazyme to the first target and to the sensor region to facilitate cleavage of the first oligonucleotide component to thereby generate the first fragment and the second fragment.

148. The method of any one of claims 57 to 147 wherein generation of the first detectable signal is not reversible at the first temperature.

149. The method of any one of claims 57 to 148 wherein the first temperature is lower than the second temperature or wherein the first temperature is higher than the second temperature.

150. The method of any one of claims 57 to 149 wherein the first enzyme does not digest the first target and / or the second target.

151. The method of any one of claims 57 to 150 wherein the first temperature differs from the second temperature by more than: 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C or 60°C.

152. The method of any one of claims 57 to 151 wherein the first temperature differs from the second temperature by at least about 16°C.

153. The method of any one of claims 57 to 152 wherein the biological sample is obtained from a subject and / or wherein the method is performed in vitro or ex vivo.

154. The method of any one of claims 57 to 153 wherein the presence or absence of the first target in a sample is determined at temperatures above the melting temperature (Tm) of the cleaved TLM probe and below the Tm of the intact TLM probe.

155. A composition comprising:a temperature-dependent looped multi-component (TLM) probe comprising a first oligonucleotide component and a second oligonucleotide component,wherein the first oligonucleotide component comprises a first capture region and a second capture region, the first capture region being capable of hybridisation to a first capture region of the second oligonucleotide component by complementary base pairing to form a first double-stranded portion, and the second capture region being capable of hybridisation to a third capture region of the first oligonucleotide component or to a second capture region of the second oligonucleotide component by complementary base pairing to form a second double-stranded portion;wherein the first oligonucleotide component is connected to a first detection moiety and the second oligonucleotide component is connected to a second detection moiety;wherein:wherein the first oligonucleotide component further comprises a single-stranded loop portion of unhybridised nucleotides comprising a sensor region capable of serving as a substrate for an enzyme, wherein the sensor region is located between the first and second capture regions of the first oligonucleotide component;wherein digestion of the sensor region by the enzyme generates a first fragment and a second fragment;wherein the first detection moiety of the first oligonucleotide is connected to the second capture region of the first oligonucleotide.

156. The composition of claim 155 wherein:the TLM probe does not comprise more than two detection moieties;the first oligonucleotide component is not connected to more than one detection moiety; or the first fragment is not directly labelled with a detection moiety.

157. The composition of claim 155 or 156, further comprising an enzyme capable of modifying the sensor region of the first oligonucleotide component only when the target is present in the sample.

158. The composition of any one of claims 155 to 157 wherein the second oligonucleotide component is not directly labelled with more than one detection moiety or wherein the second oligonucleotide component is not connected to more than one detection moiety.

159. The composition of any one of claims 155 to 157 wherein the second oligonucleotide component is directly labelled with the second detection moiety or wherein the second fragment is not directly labelled with the first detection moiety.

160. The composition of any one of claims 155 to 159 wherein the first oligonucleotide component is not directly labelled with the first detection moiety.

161. The composition of any one of claims 155 to 160 wherein the second capture region of the first oligonucleotide is capable of hybridisation to a third capture region of the first oligonucleotide component by complementary base pairing to form a second doublestranded portion, and the probe further comprises a third oligonucleotide component comprising a first capture region capable of hybridisation to a fourth capture region of the first oligonucleotide component by complementary base pairing to form a third doublestranded portion.

162. The composition of any one of claims 155 to 160 wherein the second capture region of the first oligonucleotide is capable of hybridisation to a second capture region of the second oligonucleotide component by complementary base pairing to form a second double-stranded portion, and the probe further comprises a third oligonucleotide component comprising a first capture region capable of hybridisation to a third capture region of the first oligonucleotide component by complementary base pairing to form a third double-stranded portion.

163. The composition of claim 161 or 162 wherein the first oligonucleotide component is connected to the first detection moiety via the third oligonucleotide component.

164. The composition of claim 163 wherein the first capture region differs in length or sequence from the second capture region.

165. The composition of any one of claims 155 to 159 wherein the first oligonucleotide component is directly labelled with the first detection moiety.

166. The composition of any one of claims 155 to 165 wherein:the first detection moiety is a fluorophore, and the second detection moiety is a quencher; orthe first detection moiety is a quencher, and the second detection moiety is a fluorophore.

167. The composition of any one of claims 155 to 166 wherein:the first detection moiety is a fluorophore, and the second detection moiety is a quencher; orthe first detection moiety is a quencher, and the second detection moiety is a fluorophore;and wherein the TLM probe does not comprise more than one quencher.

168. The composition of any one of claims 155 to 167 wherein the sensor region is located between the first capture region and the first detection moiety.

169. The composition of any one of claims 155 to 168 wherein following digestion of the sensor region the first fragment is capable of hybridising to the second oligonucleotide component via the first capture region.

170. The composition of any one of claims 155 to 169, further comprising a multicomponent nucleic acid enzyme (MNAzyme) comprising two partzyme oligonucleotides, each partzyme oligonucleotide having a substrate arm capable of hybridising to at least a portion of the sensor region of the first oligonucleotide component only in the presence of a target.

171. The composition of claim 170 wherein the substrate arms of the two partzyme oligonucleotides are hybridised to the sensor region of the first oligonucleotide component.

172. The composition of any one of claims 155 to 171, further comprising a DNAzyme capable of cleaving the sensor region of the first oligonucleotide component only in the presence of a target.

173. The composition of any one of claims 155 to 172, further comprising an aptazyme capable of cleaving the sensor region of the first oligonucleotide component only in the presence of a target.

174. The composition of any one of claims 155 to 173, further comprising a restriction endonuclease capable of cleaving the sensor region of the first oligonucleotide component only in the presence of a nucleic acid target.

175. The composition of any one of claims 155 to 174, further comprising an exonuclease capable of digesting the sensor region of the first oligonucleotide component only in the presence of a nucleic acid target.

176. The composition of claim 175, wherein the exonuclease is a polymerase with exonuclease activity.

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