Multiplexed detection of nucleic acids using mixed reporters

Abstract

The present invention provides oligonucleotides and methods for their use in the detection and / or differentiation of target nucleic acids. The oligonucleotides and methods find particular use in simultaneously amplifying, detecting, and / or differentiating multiple targets.

Description

[Technical Field]

[0001] 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 target nucleic acids. The oligonucleotides and methods find particular use in simultaneously amplifying, detecting, differentiating, and / or quantifying multiple targets. [Background technology]

[0002] Genetic analysis is becoming routine in the clinic for assessing disease risk, diagnosing disease, predicting patient prognosis or response to treatment, and monitoring patient progression. The introduction of such genetic testing depends on the development of simple, inexpensive, and rapid assays to identify genetic variations.

[0003] In vitro nucleic acid amplification methods have been widely applied in genetics and disease diagnosis. These methods include polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), strand displacement amplification (SDA), nicking enzyme amplification (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 (3SR), nucleic acid sequence-based amplification (NASBA), ligase chain reaction (LCR), and divergent amplification (RAM). Each of these target amplification strategies requires the use of oligonucleotide primers. The amplification process incorporates the oligonucleotide primers at their 5' ends, resulting in exponential amplification of the amplicon, which contains newly synthesized copies of the sequence located between the primers.

[0004] Commonly used methods for monitoring amplicon accumulation in real time or at the end of amplification include detection using MNAzymes with universal substrate probes, target-specific molecular beacons, sloppy beacons, eclipse probes, TaqMan or hydrolysis probes, Scorpion uniprobes or biprobes, catcher / pitcher probes, dual hybridization probes, and / or intercalating dyes such as SybGreen. Because amplicons with different sequences denature at different temperatures, known as melting temperatures or Tm, high-resolution melting curve analysis can be performed during or at the end of some of these protocols to obtain additional information. Such protocols measure melting curves resulting from either a) the separation of the two strands of a double-stranded amplicon in the presence of an intercalating dye, or b) the separation of one strand of the amplicon and a complementary target-specific probe labeled with a fluorophore and a quencher, or c) the separation of a non-target-related duplex, such as a catcher duplex that is only formed in the presence of the target. Melting curve analysis provides information about the rate of dissociation of the two DNA strands during heating. The melting temperature (Tm) is the temperature at which 50% of the DNA dissociates. Tm depends on the length, sequence composition, and GC content of the paired nucleotides. Elucidating information about the target DNA from melting curve analysis traditionally involves a series of fluorescence measurements taken at small intervals over a wide temperature range. The melting temperature does not depend only on the base sequence. It also depends on the concentration of the oligonucleotide, the cations (monovalent (Na)) in the buffer, and the amount of cations (monovalent (Na)) in the buffer. + ) and divalent (Mg 2+ ) salts), and / or the presence or absence of destabilizing agents such as urea or formamide.

[0005] Generally, the number of available fluorescence channels capable of monitoring distinct wavelengths limits the number of targets that can be detected and specifically identified in a single reaction with a fluorescence reader. Recently, a protocol known as "tagging by oligonucleotide cleavage and extension" (TOCE) has been developed. Col has expanded this capability, enabling the analysis of multiple targets at a single wavelength. TOCE technology uses pitcher and catcher oligonucleotides. The pitcher has two regions: a targeting portion that is complementary to the target, and a tagging portion located at the 5' end, which is not complementary. The capture oligonucleotide is dual-labeled and has a region at its 3' end that is complementary to the tagging portion of the pitcher. During amplification, the pitcher binds to the amplicon, and upon primer extension, the exonuclease activity of the polymerase can cleave the tagging portion from the pitcher. The released tagging portion then binds to the catcher oligonucleotide and serves as a primer for synthesizing the complementary strand. The melting temperature (catcher-Tm) of the double-stranded catcher molecule then acts as a surrogate marker for the original template. It is possible to incorporate multiple catchers with different sequences and lengths, all of which melt at different temperatures, thereby obtaining a range of catcher-Tm values ​​representing a range of targets while still measuring at a single wavelength. Limitations of this approach include inherent complexity, as the released fragments require initiation and completion of 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 associated with each specific target.

[0006] Hairpin probes or stem-loop probes have also proven to be useful tools for detecting nucleic acids and / or monitoring target amplification. One type of hairpin probe, dual-labeled with a fluorophore and quencher dye pair, is commonly known in the art as a molecular beacon. Generally, these molecules have three features: 1) a stem structure formed by hybridization of the complementary 5' and 3' ends of an oligonucleotide; 2) a loop region complementary to the target or target amplicon to be detected; and 3) a fluorophore-quencher dye pair attached to the end of the molecular beacon. During PCR, the loop region binds to the amplicon due to complementarity, which causes the stem to open and thus the fluorophore-quencher dye pair to separate. An essential feature of molecular beacons is that the loop region of these molecules remains intact during amplification and is not degraded or cleaved in the presence of the target or target amplicon. Separation of the dye pair attached to the end of the open molecular beacon causes a change in fluorescence, indicating the presence of the target. This method is commonly used for multiplex analysis of multiple targets in a single PCR test. Generally, in a multiplex assay, each molecular beacon has a different target-specific loop region and a unique fluorophore, so that the hybridization of each different molecular beacon to each amplicon species can be monitored in a separate channel, i.e., at a separate wavelength.

[0007] The concept of molecular beacons has been expanded with a strategy known as sloppy beacons. In this protocol, the loop region of a single beacon is long enough to tolerate mismatched bases and thus bind to several closely related targets that differ by one or more nucleotides. After amplification, melting curve analysis can be performed to distinguish different target species based on the temperature at which the duplex formed by hybridization between the target species and the loop region of the sloppy beacon separates (melts). In this way, multiple closely related species can be simultaneously identified by detecting them at a single wavelength and characterizing the melting profile of a specific target with a 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 drawback of DNA hybridization-based techniques such as sloppy beacons and TOCE is that they can generate false-positive results due to nonspecific hybridization between the probe and non-target nucleic acid sequences.

[0008] Many nucleic acid detection assays utilize melting curve analysis to identify the presence of a particular target sequence in a given sample or to reveal information about the amplified sequence. Melting curve analysis protocols involve measuring fluorescence at various temperatures over an increasing temperature range. The change in the slope of this curve is then plotted as a function of temperature to obtain a melting curve. This process is often slow, typically taking 30-60 minutes to complete. Furthermore, melting curve analysis may require skilled personnel and / or the use of specialized software to interpret the results. Therefore, there is a high demand for faster and / or simpler alternatives to melting curve analysis.

[0009] Melting curves are typically analyzed after PCR and therefore only allow for a qualitative determination of the presence or absence of a target in a sample. In many cases, 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 a fast alternative to melting curve analysis that also provides quantitative information about the sample.

[0010] 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. Summary of the Invention

[0011] The present invention addresses one or more of the deficiencies present in current multiplex detection assays.

[0012] Methods and compositions are provided herein that expand the ability to multiplex during amplification protocols. These methods combine "standard reporters," including substrates and probes known in the art, with structures referred to herein as LOCSs (loop-connected stems). Standard reporters include probes and substrates, including, but not limited to, linear MNAzyme substrates, TaqMan or hydrolysis probes, molecular beacons, sloppy beacons, eclipse probes, Scorpion uniprobes or biprobes, capture / pitcher oligonucleotides, and dual hybridization probes. Combining a standard reporter system with one or more LOCSs (all species can be labeled, for example, with a single detection moiety (e.g., the same fluorophore and quencher pair)) allows for the individual discrimination of multiple targets in a single reaction. This approach involves measuring the signal generated from the "standard reporter" and one or more LOCSs at one or more temperatures. The generation of a signal from a LOCS is the temperature at which the signal is measured, -Whether the loop portion of LOCS is cleaved or degraded in response to the presence of the target; - may depend on several factors, including any one or more of the melting temperatures of the stem portion of a particular LOCS in its "intact" or in its cleaved or degraded "split" conformation.

[0013] The melting temperature of the stem region of a split LOCS acts as a surrogate marker for a specific target, mediated by target-dependent cleavage or degradation of the loop of the intact LOCS. Other methods for incorporating stem-loop structures utilize a change in fluorescent signal after hybridization of the loop region to a target amplicon (e.g., molecular beacons and sloppy beacons), which increases the distance between the dye pair, or b) target-mediated cleavage, which allows for physical separation of the dyes (e.g., cleavable molecular beacons). Cleavable molecular beacons are typically used to generate positive or negative signals for a given target at a single wavelength. Multiplexed target detection generally requires detecting different targets via signals emitted at different wavelengths. Therefore, incorporating mutant stems into different cleavable molecular beacons labeled with similar or identical detection moieties and designed to detect different targets provides the ability to distinguish detectable signals representing individual targets based on differences in stem melting temperatures, without the need for different detectable signals between targets.

[0014] The present invention is based, at least in part, on the fact that each stem melts at a different temperature and generates a signal. This provides an improvement over existing multiplex detection assays that arises through manipulating the melting temperature of the stem portion of the stem-loop structure by varying the length and / or sequence composition of the stem to form a single molecule.

[0015] The present invention can involve the use of a standard reporter with a single LOCS reporter or multiple LOCS reporters in a single reaction. Both the standard and LOCS reporters can be labeled with the same or similar detection moieties that can be detected in substantially the same manner (e.g., fluorophores that emit in the same region of the visible spectrum, nanoparticles of the same size and / or type for colorimetric or SPR detection, reactive moieties (e.g., alkaline phosphatase or peroxidase enzymes) for chemiluminescent detection, or electroactive species (e.g., ferrocene, methylene blue, or peroxidase enzymes) for electrochemical detection. When multiple LOCSs are present, e.g., labeled with the same detection moiety, they can contain (a) different loop sequences, each allowing for direct or indirect simultaneous detection of multiple targets, and / or b) different stem sequences that melt at distinct temperatures and can be used to identify specific targets present in the multiple targets being interrogated. The methods of the present invention use LOCSs that offer one or more advantages over methods known in the art, such as the TOCE protocol, in that, for example, a separate catcher molecule is not required, thereby reducing the number of components in the reaction mixture and reducing costs. Furthermore, the methods of the present invention are inherently less complex than TOCE methods, which require the released fragments to initiate and complete a second extension on a synthetic target.

[0016] In some embodiments, LOCS probes can be universal (independent of the target sequence) and / or can be combined with a range of detection technologies, thus offering broad applicability in the field of molecular diagnostics. Additionally, the melting temperatures used in other conventional amplification and detection technologies are typically based on hybridization and melting of the probe by the target nucleic acid, which suffers from the drawback of increased false positives due to nonspecific hybridization between the probe and non-target nucleic acid sequences. The methods of the present invention overcome this limitation because those LOCS reporter probes containing universal substrates do not bind to the target sequence. Finally, it is well known in the art that intramolecular binding is stronger than intermolecular binding, and therefore the probability that these uncleaved (intact) LOCS will hybridize to nonspecific targets and generate false positive signals is much lower.

[0017] As a result of the intramolecular bonds being stronger than the intermolecular bonds, the dual-labeled LOCS melts at one temperature when intact and at a lower temperature after target-dependent cleavage or degradation of the loop region that splits the LOCS into two fragments. This property of nucleic acids is exploited in the present invention to expand the ability of an instrument to differentiate between multiple targets using a single type of detector, such as one fluorescent channel, or a specific mode of colorimetric, surface plasmon resonance (SPR), chemiluminescent, or electrochemical detection.

[0018] The temperature-dependent fluorescent signal generated by the LOCS reporter of the present invention is well-defined and independent of the target DNA. Therefore, it is possible to elucidate information about the target DNA from measuring the fluorescent signal generated at selected temperatures rather than a complete temperature gradient, providing an advantage in reducing run times on thermal cycling devices (e.g., PCR devices). As a non-limiting example, performing a conventional melting analysis on a Bio-Rad CFX96 PCR system using temperatures ranging from 20°C to 90°C in 0.5°C increments and a 5-second hold time requires 141 fluorescence measurement cycles and approximately 50 minutes of run time. Using LOCS probes, information about the target DNA can be obtained from the same device in 2-6 fluorescence measurements, requiring approximately 2-5 minutes of run time. Absent any specific limitations, reduced run time could be advantageous in many applications, including diagnostics.

[0019] In the present invention, LOCS probes are combined with standard reporters or probes or substrates to simultaneously detect, differentiate, and / or quantify multiple targets. Individual signals representing various targets may be detectable by the same means, for example, via signals emitted in a single fluorescent channel or by specific modes of colorimetric, surface plasmon resonance (SPR), chemiluminescent, or electrochemical detection. In traditional qPCR, target DNA quantification can be determined using cycle quantification (Cq) values ​​from an amplification curve obtained by measuring fluorescence at a single temperature in each amplification cycle. Because the Cq value is proportional to the negative logarithm of the target DNA concentration, it is possible to determine the concentration from experimentally determined Cq values. However, when two or more target-specific probes are present in a single channel, it is difficult to accurately quantify each target because it is difficult to identify which probes contribute to the signal. To address this issue, LOCS reporters can be used to generate amplification curves obtained by measuring fluorescence at two or more temperatures during amplification, enabling accurate and specific quantification of two or more targets in a single channel. This is possible because LOCS reporters can generate significantly different amounts of fluorescence at different temperatures. Furthermore, using LOCS reporters to obtain fluorescence at a first temperature (target 1) in real time and at a second temperature (target 2) before and after amplification can enable accurate and specific quantification of a first target and simultaneous qualitative detection of a second target in a single channel. The advantage of the latter scenario is that it does not affect the overall run time of the amplification protocol and may not require dedicated software for analysis. This approach may be useful in scenarios where quantification or Cq determination is required for only one of the targets.

[0020] In some embodiments where analysis only requires fluorescence acquisition at a limited number of time points within PCR, for example, at or near the beginning of amplification and post-amplification at the end point, the use of a LOCS architecture eliminates the need for acquisition at every cycle, making these embodiments well suited to very rapid cycling protocols that can reduce time to results.

[0021] As described above, melting curve analysis protocols involve measuring fluorescence at various temperatures over a gradually increasing temperature range (e.g., 30°C to 90°C). The change in slope of this curve can then be plotted as a function of temperature to obtain a melting curve. This process is often slow, taking, for example, 30 to 60 minutes to complete. Increasing the speed of melting curve analysis requires access to highly specialized equipment and cannot be achieved using standard PCR devices. Therefore, there is a high demand for faster alternatives to melting curve analysis that can provide simultaneous detection of multiple targets in a single fluorescence channel using standard equipment. The melting temperature (Tm) of the LOCS structures of the present invention is predetermined and constant (i.e., unaffected by target sequence or concentration) for given experimental conditions, eliminating the need for ramping through an entire temperature gradient. Each LOCS structure overcomes the above limitations because it requires only a single fluorescence measurement at its specific Tm, eliminating the need to run a full temperature gradient and thus facilitating faster time to results.

[0022] Furthermore, melting curve analysis typically requires interpretation by trained personnel or the use of specialized software to interpret the results.

[0023] In some embodiments of the invention, the use of fluorescence measurements at discrete temperatures after completion of PCR can eliminate the need for subjective interpretation of melting curves and facilitate objective determination of the presence or absence of a target.

[0024] In other embodiments of the invention, analysis may require only a limited number of time points within the PCR, e.g., pre-PCR and post-PCR fluorescence acquisition, which eliminates the need for acquisition at every cycle. These embodiments are therefore well suited to very rapid cycling protocols that can reduce the time to results.

[0025] Several methods have been described that involve fluorescence acquisition at multiple temperatures during PCR, including dual temperature acquisition to facilitate differentiation between perfectly matched and mismatched probes. Additionally, when two targets are present and detected from a single channel, some protocols use multiple acquisition temperatures to quantify the concentration of each target after each PCR cycle. Another method of simultaneously quantifying two targets is achieved by performing a complete melting curve at the end of each PCR cycle.

[0026] The present invention takes advantage of the benefits of combining LOCS with other types of reporter molecules. The LOCS structure may be compatible with most, and potentially all, existing methods of real-time and end-point PCR analysis. While it is possible to perform analyses using only LOCS probes to distinguish multiple targets in a single reaction, it may be advantageous to use multiple types of probes in a single reaction. By way of example, a single LOCS probe may be used in combination with any of the following technologies: linear MNAzyme substrates, linear TaqMan probes, restriction enzyme-cleavable probes, eclipse probes, non-cleavable molecular beacon probes, non-cleavable sloppy beacons, Scorpion uniprobes, Scorpion biprobes, dual hybridization probe pairs, or probes utilizing catcher and pitcher technology (e.g., TOCE probes).

[0027] In various embodiments of the invention, the single LOCS probe and linear MNAzyme substrate, linear TaqMan probe, or non-cleavable molecular beacon probe may be labeled with the same or similar detection moieties. By way of non-limiting example, this may include the same fluorophore for fluorescent detection, the same size and / or type of nanoparticle (e.g., gold or silver) for colorimetric or SPR detection, a reactive moiety (e.g., alkaline phosphatase or peroxidase enzyme) for chemiluminescent detection, or an electroactive species (e.g., ferrocene, methylene blue, or peroxidase enzyme) for electrochemical detection.

[0028] In certain embodiments, a linear MNAzyme substrate that can be cleaved by a first target-specific MNAzyme can be combined with a single LOCS probe that can be cleaved by a second target-specific MNAzyme. Embodiments in which one linear MNAzyme substrate and one LOCS probe are used to detect two targets, or at one wavelength in the visible spectrum, for example, can be advantageous over embodiments using two LOCS probes because the manufacture of the linear probe is simpler and less costly than the manufacture of the LOCS probe. This is because the linear substrate does not require the additional sequence required for the LOCS probe stem region and is therefore shorter. Similarly, the manufacture of a linear TaqMan probe can be less expensive than the manufacture of a LOCS probe.

[0029] An additional advantage of using either single or multiple LOCS probes in combination with other types of standard reporters relates to the inherent difference in background fluorescence of linear probes, where temporal / spatial parameters result in a greater distance between the fluorophore and quencher, and therefore higher background fluorescence, compared to LOCS probes in which the fluorophore and quencher are held in close proximity by a stem portion. Furthermore, different types of probes generate signals using different mechanisms and exhibit different fluorescence and quenching properties at different temperatures. In various embodiments exemplified below, this difference in fluorescence and quenching capabilities provides an additional tool that allows investigators to manipulate the magnitude of the detected signal at a specific temperature to detect, distinguish, and / or quantify multiple targets at a single wavelength. do.

[0030] In some embodiments, the invention takes advantage of the fact that LOCS probes and Catcher-Pitcher probes have opposing fluorescence / quenching properties at different temperatures. For example, regardless of the presence or absence of target, Catcher-Pitcher probes remain quenched at high temperatures (i.e., above the T of the Catcher-Pitcher duplex) due to denaturation of the duplex and conformational changes in the Catcher strand. Conversely, regardless of the presence or absence of target, LOCS probes remain quenched at low temperatures (i.e., below the T of the split LOCS stem) because the hybridized stem holds the fluorophore and quencher in close proximity. Furthermore, in the presence of target, Catcher-Pitcher probes generate an increase in fluorescence at low temperatures (i.e., below the T of the Catcher-Pitcher duplex), while LOCS probes generate an increase in fluorescence at high temperatures (i.e., above the T of the split LOCS stem). These opposing fluorescence / quenching properties at high and low temperatures allow for the specific detection of two targets: one target can be detected at a first, lower temperature using the Catcher-Pitcher probe, and another target can be detected at a second, higher temperature using the LOCS probe.

[0031] Various embodiments of the present invention take advantage of the advantages of combining a single linear substrate or probe, such as a linear MNAzyme substrate or TaqMan probe, with a LOCS probe. For example, compared to using a pair of LOCS probes with one low-Tm stem and one high-Tm stem, the advantage is that both the cleaved linear MNAzyme substrate and the degraded TaqMan probe generate similar fluorescent signals over a wide temperature range. Similarly, the uncleaved linear MNAzyme substrate and the intact TaqMan probe generate similar fluorescent signals over a wide temperature range. Thus, for both probe types, the signal-to-noise ratio is constant over a wide range of detection temperatures. In comparison, the observed signal-to-noise ratio resulting from a split low-Tm LOCS probe may decrease at higher detection temperatures due to the greater background fluorescence generated by the denaturation of the intact LOCS stem. This means that cleavage of a linear MNAzyme substrate or TaqMan probe can be detected over a wider detection temperature range compared to a low-Tm LOCS probe, which has a more limited detection temperature range. This allows for greater flexibility in thermal cycling and may be useful for the development of faster and simplified multiplex assays. A further advantage comes from the ability to combine one or more LOCS probes with existing commercial kits using other technologies, such as TaqMan probes, thus expanding their multiplexing capabilities.

[0032] In other embodiments, the present invention takes advantage of the fact that LOCS probes and Scorpion uniprobes or biprobes also behave differently at different temperatures, enabling specific detection of two targets at two different detection temperatures. For example, at a high detection temperature, if the stem is open and fluorescent and the loop cannot bind to the amplicon of a specific target (target 1), the Scorpion uniprobe can always be fluorescent (pre-PCR and post-PCR), regardless of the presence or absence of either target. Similarly, at a high detection temperature, the complementary quencher sequence cannot bind to the probe, potentially preventing the probe from binding to the amplicon of the specific target (target 1), so the Scorpion biprobe can always be fluorescent (pre-PCR and post-PCR), regardless of the presence or absence of either target. In both cases (uniprobe or biprobe), at the same high temperature, the LOCS probe will only produce fluorescence in the presence of the specific target (target 2) due to cleavage and dissociation of the stem. Conversely, at low detection temperatures, the LOCS probe is always quenched (pre- and post-PCR) regardless of the presence or absence of either target because the Tm of both the intact LOCS and split-LOCS stems is higher than this temperature. Meanwhile, at this same temperature, the Scorpion uniprobe or biprobe generates fluorescence only in the presence of a specific target due to hybridization of the loop or probe region, respectively, to the target 1 amplicon. These opposing fluorescence / quenching properties at high and low temperatures allow for the specific detection of two targets: target 1 can be detected at a first, lower temperature using either the Scorpion uniprobe or Scorpion biprobe, and target 2 can be detected at a second, higher temperature using the LOCS probe.

[0033] Various types of standard reporter substrates and probes fluoresce over a wide range of temperatures or only over a limited range. For example, linear reporter substrates or probes, including but not limited to linear MNAzyme substrates, eclipse probes, TaqMan probes, hydrolysis probes, and others, generally generate a fluorescent signal over a wide range of temperatures. Such probes are generally quenched before PCR and fluoresce after PCR if the target is present, and this fluorescence can be measured over a wide range of temperatures. In contrast, LOCS probes, molecular beacons, Scorpion uniprobes or biprobes, and pitcher and catcher fluorescence systems (e.g., TOCE probes) can be engineered so that they are fluorescent or quenched within a defined temperature range.

[0034] Molecular beacons are quenched by hybridized stems at temperatures below the temperature at which the molecular beacon loop binds to the target and fluoresces. In contrast, the stem of an intact LOCS probe is hybridized at a temperature above the temperature at which the split LOCS probe melts. Furthermore, while many reporter systems measure an increase in fluorescence in the presence of a target, other techniques, such as dual hybridization probes, result in a decrease in fluorescence when the target is present. The present invention provides a new method for combining probes and setting parameters so that specific probe combinations can be used to increase or decrease the detectable signal at specific temperatures, enabling improved multiplexing scenarios. Thus, the use of different types of standard reporter substrates and the differential behavior of probes allows for the manipulation of the presence or absence of a signal, such as fluorescence or quenching, at multiple temperatures when combined with LOCS probes labeled with the same or similar detection moieties and present in a single reaction, which in turn provides numerous advantages for target analysis.

[0035] The invention relates, at least in part, to the following embodiments 1-194.

[0036] Embodiment 1. A method for determining the presence or absence of a first and a second target in a sample, comprising: (a) a sample or a derivative thereof suspected of containing a first and a second target; a first oligonucleotide for the detection of a first target, the first oligonucleotide comprising a first detection moiety capable of generating a first detectable signal; an intact stem-loop oligonucleotide for the detection of a second target, comprising a double-stranded stem portion of hybridized nucleotides, the opposite strand of which is connected by an unbroken single-stranded loop portion of unhybridized nucleotides, the stem portion comprising a second detection moiety capable of generating a second detectable signal; the first and second detection moieties are intact stem-loop oligonucleotides capable of producing indistinguishable detectable signals at a single temperature using a single type of detector; and - preparing a reaction mixture by contacting with a first enzyme capable of digesting one or more unhybridized nucleotides of the intact stem-loop oligonucleotide only if a second target is present in the sample; (b) adding the mixture to - the first target is suitable for inducing a modification to the first oligonucleotide, thereby enabling the first detection moiety to generate a first detectable signal; - treating under conditions suitable for digesting one or more of the unhybridized nucleotides of the intact stem-loop oligonucleotide with a first enzyme only if the second target is present in the sample, thereby disrupting the single-stranded loop portion and providing a split stem-loop oligonucleotide; (c) - measuring the background signal provided by the first and second detection moieties in the mixture or in a control mixture; (d) at one or more points during or after such processing: - whether a first detectable signal resulting from the modification is produced at the first temperature, the first detectable signal being distinct from background signal and indicative of the presence of the first target in the sample; - determining whether a second detectable signal is produced at the second temperature that is distinct from the background signal and that indicates the presence of a second target in the sample; at the first temperature, the second detectable signal is not different from the background signal; At the second temperature, if present, the strands of the double-stranded stem portion of the split stem-loop oligonucleotide are partially or completely dissociated, allowing the second detection moiety to provide a second detectable signal; When present, the strands of the double-stranded stem portion of the intact stem-loop oligonucleotide cannot dissociate, thereby preventing the second detectable moiety from providing a second detectable signal.

[0037] Embodiment 2. The determination in part (d) is: - using a predetermined threshold value to determine whether a first detectable signal resulting from said modification differs from any said background signal at a first temperature; and / or 2. The method of embodiment 1, comprising using a predetermined threshold value to determine whether the second detectable signal is different from any relevant background signal at the second temperature.

[0038] Embodiment 3. The control mixture comprises: - the primary target, or - a secondary target, or - Primary and secondary targets 3. The method of claim 1 or 2, wherein the method does not comprise the steps of:

[0039] Embodiment 4. The control mixture comprises a predetermined amount of - the primary target, or - a secondary target, or - Primary and secondary targets 4. The method of any one of embodiments 1 to 3, comprising the steps of:

[0040] Embodiment 5. - a modification to the first oligonucleotide enables the first detection moiety to provide a first detectable signal at or below a first temperature; The method according to any one of embodiments 1 to 4, wherein the generation of the first detectable signal is reversible.

[0041] Embodiment 6. -Part (c) is measuring a first background signal within 1°C, 2°C, 3°C, 4°C, or 5°C of a first temperature and a second background signal within 1°C, 2°C, 3°C, 4°C, or 5°C of a second temperature; These are provided by first and second detection moieties in the mixture or in the control mixture, - Part (d) at one or more times during or after such processing: whether a first detectable signal resulting from the modification is produced at the first temperature that is distinct from a first background signal and is indicative of the presence of the first target in the sample; 6. The method of embodiment 5, comprising determining whether a second detectable signal is produced at the second temperature that is distinct from a second background signal and that indicates the presence of a second target in the sample.

[0042] Embodiment 7. - the first target is a nucleic acid sequence; - the first oligonucleotide is a stem-loop oligonucleotide comprising a double-stranded stem portion of hybridized nucleotides on opposite strands connected by an unbroken single-stranded loop portion of unhybridized nucleotides, all or part of which is complementary to the first target; 7. The method of embodiment 5 or 6, wherein the modification of the first oligonucleotide is a conformational change resulting from hybridization of the target to the single-stranded loop portion of the first oligonucleotide by complementary base pairing.

[0043] Embodiment 8. 8. The method of embodiment 7, wherein the conformational change is strand separation of the double-stranded stem portion of the first oligonucleotide resulting from said hybridization of the target to the single-stranded loop portion of the first oligonucleotide by complementary base pairing.

[0044] Embodiment 9. - the stem portion of the first oligonucleotide has a melting temperature (Tm) that is less than the Tm of the double-stranded duplex structure formed from said hybridization of the target to the single-stranded loop portion of the first oligonucleotide and greater than the Tm of the stem portion of the split stem-loop oligonucleotide; - the double-stranded duplex structure has a Tm that is greater than the Tm of the stem portion of the intact stem-loop oligonucleotide and greater than the Tm of the stem portion of the split stem-loop oligonucleotide; - the stem portion of the intact stem-loop oligonucleotide has a Tm that is greater than the Tm of the stem portion of the split stem-loop oligonucleotide; the first temperature is below the Tm of the double-stranded duplex structure, the stem portion of the first oligonucleotide, 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 double-stranded duplex structure, the stem portion of the first oligonucleotide, and the stem portion of the intact stem-loop oligonucleotide, and above the Tm of the stem portion of the split stem-loop oligonucleotide; The method of embodiment 7 or embodiment 8, wherein the first temperature is less than the second temperature.

[0045] Embodiment 10. - the stem portion of the first oligonucleotide has a melting temperature (Tm) that is below the Tm of the double-stranded duplex structure formed from said hybridization of the target to the single-stranded loop portion of the first oligonucleotide and is below the Tm of the stem portion of the intact stem-loop oligonucleotide; - the double-stranded duplex structure has a Tm has a Tm greater than - the stem portion of the intact stem-loop oligonucleotide has a Tm that is greater than the Tm of the stem portion of the split stem-loop oligonucleotide; the first temperature is below the Tm of the double-stranded duplex structure, the stem portion of the first oligonucleotide, the stem portion of the intact stem-loop oligonucleotide, and the stem portion of the split stem-loop oligonucleotide; the second temperature is above the Tm of the stem portion of the first oligonucleotide and the stem portion of the split stem-loop oligonucleotide, and below the Tm of the double-stranded duplex structure and the stem portion of the intact stem-loop oligonucleotide; The method of embodiment 7 or embodiment 8, wherein the first temperature is less than the second temperature.

[0046] Embodiment 11. - the stem portion of the first oligonucleotide has a melting temperature (Tm) that is less than the Tm of the double-stranded duplex structure formed from said hybridization of the target to the single-stranded loop portion of the first oligonucleotide, less than the Tm of the stem portion of the intact stem-loop oligonucleotide, and less than the Tm of the stem portion of the split stem-loop oligonucleotide; - the double-stranded duplex structure has a Tm that is less than the Tm of the stem portion of the intact stem-loop oligonucleotide; - the stem portion of the intact stem-loop oligonucleotide has a Tm that is greater than the Tm of the stem portion of the split stem-loop oligonucleotide; the first temperature is below the Tm of the double-stranded duplex structure, the stem portion of the first oligonucleotide, the stem portion of the intact stem-loop oligonucleotide, and the stem portion of the split stem-loop oligonucleotide; the second temperature is above the Tm of the double-stranded duplex structure, the stem portion of the first oligonucleotide, and the stem portion of the split stem-loop oligonucleotide, and below the Tm of the stem portion of the intact stem-loop oligonucleotide; The method of embodiment 7 or embodiment 8, wherein the first temperature is less than the second temperature.

[0047] Embodiment 12. - the stem portion of the first oligonucleotide has a melting temperature (Tm) that is less than the Tm of the double-stranded duplex structure formed from said hybridization to the single-stranded loop portion of the target first oligonucleotide, greater than the Tm of the stem portion of the intact stem-loop oligonucleotide, and greater than the Tm of the stem portion of the split stem-loop oligonucleotide; - the double-stranded duplex structure has a Tm that is greater than the Tm of the stem portion of the intact stem-loop oligonucleotide and greater than the Tm of the stem portion of the split stem-loop oligonucleotide; - the stem portion of the intact stem-loop oligonucleotide has a Tm that is greater than 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 first oligonucleotide and the double-stranded duplex structure and above 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 first oligonucleotide, the double-stranded duplex structure, and the stem portion of the intact stem-loop oligonucleotide, and above the Tm of the stem portion of the split stem-loop oligonucleotide; The method of embodiment 7 or embodiment 8, wherein the first temperature is greater than the second temperature.

[0048] Embodiment 13. - the Tm of the stem portion of the first oligonucleotide is 1°C to 10°C, 1°C to 5°C, 5°C to 10°C, or more than 10°C lower than the Tm of the double-stranded duplex structure; and / or the Tm of the stem portion of the intact stem-loop oligonucleotide is 1°C to 10°C, 1°C to 5°C, 5°C to 10°C, or lower than the Tm of the stem portion of the split stem-loop oligonucleotide; or more than 10°C higher, and / or the first temperature is 1°C to 10°C, 1°C to 5°C, 5°C to 10°C, or more than 10°C lower than the Tm of the stem portion of the first oligonucleotide and / or the double-stranded duplex structure; and / or the second temperature is between 1°C and 10°C, between 1°C and 5°C, between 5°C and 10°C, or more than 10°C lower than the Tm of the stem portion of the intact stem-loop oligonucleotide; and / or - The method of any one of embodiments 7 to 12, wherein the second temperature is 1°C to 10°C, 1°C to 5°C, 5°C to 10°C, or more than 10°C higher than the Tm of the stem portion of the split stem-loop oligonucleotide.

[0049] Embodiment 14. - the first target is a nucleic acid sequence; the first oligonucleotide is a double-stranded stem portion of hybridized nucleotides, the opposing strands of which are connected by a single-stranded loop portion of unhybridized nucleotides, all or a portion of which is complementary to a first target, and a second single-stranded portion extending in the 3' direction from one of the opposing strands and terminating in a sequence complementary to a portion of the first target; a stem-loop oligonucleotide comprising a blocker molecule preceding a sequence complementary to a portion of a first target; the mixture further comprises a polymerase; - treating the mixture hybridizing the second single-stranded portion to the first target by complementary base pairing; extending a second single-stranded portion using a polymerase and the first target as a template sequence to provide a double-stranded nucleic acid, wherein the blocker molecule prevents the polymerase from using the stem portion of the first oligonucleotide as a template to extend the first target; 7. The method of embodiment 5 or embodiment 6, comprising: denaturing the double-stranded nucleic acid and providing a modification to the first oligonucleotide that enables the second single-stranded portion extended by the polymerase to hybridize to the single-stranded loop portion of the first oligonucleotide by complementary base pairing to generate a signaling duplex structure, thereby enabling the first detection moiety to provide a first detectable signal.

[0050] Embodiment 15. - the stem portion of the first oligonucleotide has a melting temperature (Tm) that is less than the Tm of the signaling duplex and greater than the Tm of the stem portion of the split stem-loop oligonucleotide; the signaling duplex has a Tm that is greater than the Tm of the stem portion of the intact stem-loop oligonucleotide and greater than the Tm of the stem portion of the split stem-loop oligonucleotide; - the stem portion of the intact stem-loop oligonucleotide has a Tm that is greater than the Tm of the stem portion of the split stem-loop oligonucleotide; the first temperature is below the Tm of the signaling duplex structure, the stem portion of the first oligonucleotide, 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 signaling duplex, the stem portion of the first oligonucleotide, and the stem portion of the intact stem-loop oligonucleotide, and above the Tm of the stem portion of the split stem-loop oligonucleotide; 15. The method of embodiment 14, wherein the first temperature is less than the second temperature.

[0051] Embodiment 16. - the stem portion of the first oligonucleotide has a melting temperature (Tm) that is less than the Tm of the signaling duplex and less than the Tm of the stem portion of the intact stem-loop oligonucleotide; the signaling duplex has a Tm that is greater than the Tm of the stem portion of the split stem-loop oligonucleotide; - the stem portion of the intact stem-loop oligonucleotide has a Tm that is greater than the Tm of the stem portion of the split stem-loop oligonucleotide; the first temperature is below the Tm of the signaling duplex structure, the stem portion of the first oligonucleotide, the stem portion of the intact stem-loop oligonucleotide, and the stem portion of the split stem-loop oligonucleotide; the second temperature is above the Tm of the stem portion of the first oligonucleotide and the stem portion of the split stem-loop oligonucleotide, and below the Tm of the signaling duplex and the stem portion of the intact stem-loop oligonucleotide; 15. The method of embodiment 14, wherein the first temperature is less than the second temperature.

[0052] Embodiment 17. - the stem portion of the first oligonucleotide has a melting temperature (Tm) that is less than the Tm of the signaling duplex, less than the Tm of the stem portion of the intact stem-loop oligonucleotide, and less than the Tm of the stem portion of the split stem-loop oligonucleotide; the signaling duplex has a Tm that is less than the Tm of the stem portion of the intact stem-loop oligonucleotide; - the stem portion of the intact stem-loop oligonucleotide has a Tm that is greater than the Tm of the stem portion of the split stem-loop oligonucleotide; the first temperature is below the Tm of the signaling duplex structure, the stem portion of the first oligonucleotide, the stem portion of the intact stem-loop oligonucleotide, and the stem portion of the split stem-loop oligonucleotide; the second temperature is above the Tm of the signaling duplex, the stem portion of the first oligonucleotide, and the stem portion of the split stem-loop oligonucleotide, and below the Tm of the stem portion of the intact stem-loop oligonucleotide; 15. The method of embodiment 14, wherein the first temperature is less than the second temperature.

[0053] Embodiment 18. - the stem portion of the first oligonucleotide has a melting temperature (Tm) that is less than the Tm of the signaling duplex, greater than the Tm of the stem portion of the intact stem-loop oligonucleotide, and greater than the Tm of the stem portion of the split stem-loop oligonucleotide; the signaling duplex has a Tm that is greater than the Tm of the stem portion of the intact stem-loop oligonucleotide and greater than the Tm of the stem portion of the split stem-loop oligonucleotide; - the stem portion of the intact stem-loop oligonucleotide has a Tm that is greater than 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 first oligonucleotide and the signaling duplex and above 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 first oligonucleotide, the signaling duplex, and the stem portion of the intact stem-loop oligonucleotide, and above the Tm of the stem portion of the split stem-loop oligonucleotide; 15. The method of embodiment 14, wherein the first temperature is greater than the second temperature.

[0054] Embodiment 19. - the Tm of the stem portion of the first oligonucleotide is 1°C to 10°C, 1°C to 5°C, 5°C to 10°C, or more than 10°C lower than the Tm of the signaling duplex; and / or - the Tm of the stem portion of the intact stem-loop oligonucleotide is 1°C to 10°C, 1°C to 5°C, 5°C to 10°C, or more than 10°C higher than the Tm of the stem portion of the split stem-loop oligonucleotide; and / or - the first temperature is between 1°C and 10°C, between 1°C and 5°C, between 5°C and 10°C, or more than 10°C below the Tm of the stem portion of the first oligonucleotide and / or the signaling duplex; and / or the second temperature is between 1°C and 10°C, between 1°C and 5°C, between 5°C and 10°C, or more than 10°C lower than the Tm of the stem portion of the intact stem-loop oligonucleotide; and / or - The method of any one of embodiments 14 to 18, wherein the second temperature is 1°C to 10°C, 1°C to 5°C, 5°C to 10°C, or more than 10°C higher than the Tm of the stem portion of the split stem-loop oligonucleotide.

[0055] Embodiment 20. - The method according to any one of embodiments 5 to 19, wherein the first detection moiety is a fluorophore and the modification increases its distance from the quencher molecule.

[0056] Embodiment 21. 21. The method of embodiment 20, wherein the first oligonucleotide comprises a quencher molecule.

[0057] Embodiment 22. 22. The method of embodiment 21, wherein the fluorophore and quencher molecules are located on opposite strands of the double-stranded stem portion of the first oligonucleotide.

[0058] Embodiment 23. - the first target is a nucleic acid sequence; the first oligonucleotide is a first double-stranded portion of hybridized nucleotides, the first strand extending to a single-stranded portion terminating in a complementary sequence capable of hybridizing to a portion of a first target, the first strand including a blocker molecule preceding the complementary sequence; the mixture further comprises a polymerase; - treating the mixture hybridizing the complementary sequence of the single-stranded portion to a portion of the first target by complementary base pairing; extending a complementary sequence using a polymerase and the first target as a template sequence to provide a second double-stranded portion, wherein the blocker molecule prevents the polymerase from using the first strand of the first double-stranded portion as a template to extend the first target; denaturing the first and second double-stranded portions; providing a modification to the first oligonucleotide that hybridizes, by complementary base pairing, with a complementary sequence extended by the polymerase onto the first strand of the first double-stranded portion to generate a signaling duplex structure, thereby enabling the first detection moiety to provide a first detectable signal.

[0059] Embodiment 24. - the first double-stranded portion has a melting temperature (Tm) that is less than the Tm of the signaling duplex structure and greater than the Tm of the stem portion of the split stem-loop oligonucleotide; the signaling duplex has a Tm that is greater than the Tm of the stem portion of the intact stem-loop oligonucleotide and greater than the Tm of the stem portion of the split stem-loop oligonucleotide; - the stem portion of the intact stem-loop oligonucleotide has a Tm that is greater than the Tm of the stem portion of the split stem-loop oligonucleotide; the first temperature is below the Tm of the signaling duplex structure, the first double-stranded portion, 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 signaling duplex structure, the first double-stranded portion, and the stem portion of the intact stem-loop oligonucleotide, and above the Tm of the stem portion of the split stem-loop oligonucleotide; 24. The method of embodiment 23, wherein the first temperature is less than the second temperature.

[0060] Embodiment 25. - the first double-stranded portion has a melting temperature (Tm) that is less than the Tm of the signaling duplex structure and less than the Tm of the stem portion of the intact stem-loop oligonucleotide; the signaling duplex has a Tm that is greater than the Tm of the stem portion of the split stem-loop oligonucleotide; - the stem portion of the intact stem-loop oligonucleotide has a Tm that is greater than the Tm of the stem portion of the split stem-loop oligonucleotide; the first temperature is below the Tm of the signaling duplex structure, the first double-stranded portion, the stem portion of the intact stem-loop oligonucleotide, and the stem portion of the split stem-loop oligonucleotide; the second temperature is above the Tm of the first duplex portion and the stem portion of the split stem-loop oligonucleotide and below the Tm of the signaling duplex structure and the stem portion of the intact stem-loop oligonucleotide; 24. The method of embodiment 23, wherein the first temperature is less than the second temperature.

[0061] Embodiment 26. - the first double-stranded portion has a melting temperature (Tm) that is less than the Tm of the signaling duplex structure, less than the Tm of the stem portion of the intact stem-loop oligonucleotide, and less than the Tm of the stem portion of the split stem-loop oligonucleotide; the signaling duplex has a Tm that is less than the Tm of the stem portion of the intact stem-loop oligonucleotide; - the stem portion of the intact stem-loop oligonucleotide has a Tm that is greater than the Tm of the stem portion of the split stem-loop oligonucleotide; the first temperature is below the Tm of the signaling duplex structure, the first double-stranded portion, the stem portion of the intact stem-loop oligonucleotide, and the stem portion of the split stem-loop oligonucleotide; the second temperature is above the Tm of the signaling duplex structure, the first double-stranded portion, and the stem portion of the split stem-loop oligonucleotide, and below the Tm of the stem portion of the intact stem-loop oligonucleotide; 24. The method of embodiment 23, wherein the first temperature is less than the second temperature.

[0062] Embodiment 27. - the first double-stranded portion has a melting temperature (Tm) that is less than the Tm of the signaling duplex structure, greater than the Tm of the stem portion of the intact stem-loop oligonucleotide, and greater than the Tm of the stem portion of the split stem-loop oligonucleotide; the signaling duplex has a Tm that is greater than the Tm of the stem portion of the intact stem-loop oligonucleotide and greater than the Tm of the stem portion of the split stem-loop oligonucleotide; - the stem portion of the intact stem-loop oligonucleotide has a Tm that is greater than the Tm of the stem portion of the split stem-loop oligonucleotide; - the first temperature is below the Tm of the first double-stranded portion and the signaling duplex structure and above 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 first double-stranded portion, the signaling duplex structure, and the stem portion of the intact stem-loop oligonucleotide, and above the Tm of the stem portion of the split stem-loop oligonucleotide; 24. The method of embodiment 23, wherein the first temperature is greater than the second temperature.

[0063] Embodiment 28. - the Tm of the first double-stranded portion is 1°C to 10°C, 1°C to 5°C, 5°C to 10°C, or more than 10°C lower than the Tm of the signaling duplex; and / or - the Tm of the stem portion of the intact stem-loop oligonucleotide is 1°C to 10°C, 1°C to 5°C, 5°C to 10°C, or more than 10°C higher than the Tm of the stem portion of the split stem-loop oligonucleotide; and / or - the first temperature is between 1°C and 10°C, between 1°C and 5°C, between 5°C and 10°C, or more than 10°C below the Tm of the first double-stranded portion and / or the signaling duplex; and / or the second temperature is between 1°C and 10°C, between 1°C and 5°C, between 5°C and 10°C, or more than 10°C lower than the Tm of the stem portion of the intact stem-loop oligonucleotide; and / or - The method of any one of embodiments 23 to 27, wherein the second temperature is 1°C to 10°C, 1°C to 5°C, 5°C to 10°C, or more than 10°C higher than the Tm of the stem portion of the split stem-loop oligonucleotide.

[0064] Embodiment 29. The method according to any one of embodiments 23 to 28, wherein the first detection moiety is a fluorophore and the modification increases its distance from the quencher molecule.

[0065] Embodiment 30. 30. The method of embodiment 29, wherein the first oligonucleotide comprises a quencher molecule.

[0066] Embodiment 31. 31. The method of embodiment 30, wherein the fluorophore and quencher molecules are located on opposite strands of the first double-stranded portion.

[0067] Embodiment 32. - the first target is a nucleic acid sequence; The mixture is a first primer complementary to a first sequence in a first target; a second oligonucleotide comprising a component complementary to a second sequence in the first target that is different from the first sequence, and a tag portion that is not complementary to the first target; a first polymerase comprising an exonuclease activity; and optionally a second polymerase; - treating the mixture conditions suitable for hybridizing the first primer and the second oligonucleotide to the first target; extending the first primer using a first polymerase and the target as a template, thereby cleaving the tag portion; hybridizing the cleaved tag portion to a first oligonucleotide by complementary base pairing; using the first or second polymerase and the first oligonucleotide as a template 7. The method of embodiment 5 or embodiment 6, comprising extending the tag portion to generate a double-stranded sequence comprising the first oligonucleotide, thereby providing the modification to the first oligonucleotide and enabling the first detection moiety to provide the first detectable signal.

[0068] Embodiment 33. - the double-stranded sequence has a Tm that is greater than the Tm of the stem portion of the intact stem-loop oligonucleotide and greater than the Tm of the stem portion of the split stem-loop oligonucleotide; - the stem portion of the intact stem-loop oligonucleotide has a Tm that is greater than the Tm of the stem portion of the split stem-loop oligonucleotide; - the first temperature is below the Tm of the double-stranded sequence, 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 double-stranded sequence and the stem portion of the intact stem-loop oligonucleotide and above the Tm of the stem portion of the split stem-loop oligonucleotide; 33. The method of embodiment 32, wherein the first temperature is less than the second temperature.

[0069] Embodiment 34. - the double-stranded sequence has a Tm that is greater than the Tm of the stem portion of the split stem-loop oligonucleotide; - the stem portion of the intact stem-loop oligonucleotide has a Tm that is greater than the Tm of the stem portion of the split stem-loop oligonucleotide; - the first temperature is below the Tm of the double-stranded sequence, the stem portion of the intact stem-loop oligonucleotide, and the stem portion of the split stem-loop oligonucleotide; the second temperature is greater than the Tm of the stem portion of the split stem-loop oligonucleotide and less than the Tm of the double-stranded sequence and the stem portion of the intact stem-loop oligonucleotide; 33. The method of embodiment 32, wherein the first temperature is less than the second temperature.

[0070] Embodiment 35. - the double-stranded sequence has a Tm that is less than the Tm of the stem portion of the intact stem-loop oligonucleotide; - the stem portion of the intact stem-loop oligonucleotide has a Tm that is greater than the Tm of the stem portion of the split stem-loop oligonucleotide; - the first temperature is below the Tm of the double-stranded sequence, the stem portion of the intact stem-loop oligonucleotide, and the stem portion of the split stem-loop oligonucleotide; - the second temperature is above the Tm of the double-stranded sequence and the stem portion of the split stem-loop oligonucleotide, and below the Tm of the stem portion of the intact stem-loop oligonucleotide; 33. The method of embodiment 32, wherein the first temperature is less than the second temperature.

[0071] Embodiment 36. - the double-stranded sequence has a Tm that is greater than the Tm of the stem portion of the intact stem-loop oligonucleotide and greater than the Tm of the stem portion of the split stem-loop oligonucleotide; - the stem portion of the intact stem-loop oligonucleotide has a Tm that is greater than the Tm of the stem portion of the split stem-loop oligonucleotide; - the first temperature is below the Tm of the double-stranded sequence and above 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 double-stranded sequence and the stem portion of the intact stem-loop oligonucleotide and above the Tm of the stem portion of the split stem-loop oligonucleotide; 33. The method of embodiment 32, wherein the first temperature is greater than the second temperature.

[0072] Embodiment 37. - the Tm of the stem portion of the intact stem-loop oligonucleotide is 1°C to 10°C, 1°C to 5°C, 5°C to 10°C, or more than 10°C higher than the Tm of the stem portion of the split stem-loop oligonucleotide; and / or - the first temperature is between 1°C and 10°C, between 1°C and 5°C, between 5°C and 10°C, or more than 10°C below the Tm of the double-stranded sequence; and / or the second temperature is between 1°C and 10°C, between 1°C and 5°C, between 5°C and 10°C, or more than 10°C lower than the Tm of the stem portion of the intact stem-loop oligonucleotide; and / or - The method of any one of embodiments 32 to 36, wherein the second temperature is 1°C to 10°C, 1°C to 5°C, 5°C to 10°C, or more than 10°C higher than the Tm of the stem portion of the split stem-loop oligonucleotide.

[0073] Embodiment 38. the first oligonucleotide comprises a fluorophore and a quencher molecule; - The method according to any one of embodiments 32 to 37, wherein extending the tag moiety increases the distance between the fluorophore and the quencher molecule.

[0074] Embodiment 39. - the first target is a nucleic acid sequence; - the first oligonucleotide is complementary to a first portion of the target; the mixture further comprises an additional oligonucleotide complementary to a second portion of the first target, wherein the first and second portions of the first target are adjacent to but do not overlap one another; - treating the mixture (i) a first double-stranded component by hybridizing a first oligonucleotide to a target by complementary base pairing; and (ii) hybridizing an additional oligonucleotide to the target by complementary base pairing to form a duplex structure comprising a second double-stranded component; 7. The method of embodiment 5 or embodiment 6, comprising providing said modification to the first oligonucleotide, thereby bringing the first and further oligonucleotides into proximity and enabling the first detection moiety to provide a first detectable signal.

[0075] Embodiment 40. - the duplex structure has a Tm that is less than the Tm of the stem portion of the intact stem-loop oligonucleotide; - the stem portion of the intact stem-loop oligonucleotide has a Tm that is greater than the Tm of the stem portion of the split stem-loop oligonucleotide; - the first temperature is below the Tm of the duplex structure, the stem portion of the intact stem-loop oligonucleotide, and the stem portion of the split stem-loop oligonucleotide; - the second temperature is above the Tm of the duplex structure and the stem portion of the split stem-loop oligonucleotide and below the Tm of the stem portion of the intact stem-loop oligonucleotide; 40. The method of embodiment 39, wherein the first temperature is less than the second temperature.

[0076] Embodiment 41. - the Tm of the stem portion of the intact stem-loop oligonucleotide is 1°C to 10°C, 1°C to 5°C, 5°C to 10°C, or more than 10°C higher than the Tm of the stem portion of the split stem-loop oligonucleotide; and / or - the first temperature is between 1°C and 10°C, between 1°C and 5°C, between 5°C and 10°C, or more than 10°C below the Tm of the duplex structure; and / or the second temperature is between 1°C and 10°C, between 1°C and 5°C, between 5°C and 10°C, or more than 10°C lower than the Tm of the stem portion of the intact stem-loop oligonucleotide; and / or - the second temperature is 1°C higher than the Tm of the stem portion of the split stem-loop oligonucleotide; The method of embodiment 39 of embodiment 40, wherein the temperature is higher by up to 10°C, 1°C to 5°C, 5°C to 10°C, or more than 10°C.

[0077] Embodiment 42. - the first detectable moiety is a fluorophore and the further oligonucleotide comprises a quencher; forming the bilayer structure further brings the fluorophore and quencher into close proximity; The method according to any one of embodiments 39 to 41, wherein said detectable signal is a decrease in fluorescence provided by the first detection moiety.

[0078] Embodiment 43. - the first target is a nucleic acid sequence; the first detection moiety is a nanoparticle, a metal nanoparticle, a noble metal nanoparticle, an alkali metal nanoparticle, a gold nanoparticle, or a silver nanoparticle having a first oligonucleotide attached thereto; - treating the mixture hybridizing the first target to the first oligonucleotide, thereby introducing a modification to the first oligonucleotide that enables the first detection moiety to provide a first detectable signal indicative of the presence of the first target in the sample; the first detectable signal arises from the first detection moiety after said modification of the first oligonucleotide; (i) a change in refractive index, (ii) a change in color, and / or (iii) Changes in absorption spectrum 7. The method of embodiment 5 or embodiment 6, wherein

[0079] Embodiment 44. - the first target is a nucleic acid sequence; the first detection moiety is an electrochemical agent to which a first oligonucleotide is attached; - treating the mixture hybridizing the first target to the first oligonucleotide, thereby inducing or promoting a modification to the first oligonucleotide that enables the first detection moiety to provide a first detectable signal indicative of the presence of the first target in the sample; 7. The method of embodiment 5 or embodiment 6, wherein the first detectable signal is a change in electrochemical signal resulting from the first detection moiety after said modification of the first oligonucleotide.

[0080] Embodiment 45. 45. The method of embodiment 44, wherein the electrochemical agent is selected from any one or more of nanoparticles, methylene blue, toluene blue, oracet blue, Hoechst 33258, [Ru(phen)3]2+, ferrocene, and / or daunomycin.

[0081] Embodiment 46. the first detection moiety is a nanoparticle, a metal nanoparticle, a noble metal nanoparticle, an alkali metal nanoparticle, a gold nanoparticle, or a silver nanoparticle having a first oligonucleotide attached thereto; - the first detectable signal arises from the first detection moiety after said modification of the first oligonucleotide; (i) a change in refractive index, (ii) a change in color, and / or (iii) Changes in absorption spectrum 42. The method of any one of embodiments 5 to 19, 23 to 28, 32 to 37, and 39 to 41, wherein

[0082] Embodiment 47. the first detection moiety is an electrochemical agent to which a first oligonucleotide is attached; The method according to any one of embodiments 5 to 19, 23 to 28, 32 to 37, and 39 to 41, wherein the first detectable signal is a change in the electrochemical signal resulting from the first detection moiety after said modification of the first oligonucleotide.

[0083] Embodiment 48. 48. The method of embodiment 47, wherein the electrochemical agent is selected from any one or more of nanoparticles, methylene blue, toluene blue, oracet blue, Hoechst 33258, [Ru(phen)3]2+, ferrocene, and / or daunomycin.

[0084] Embodiment 49. the second detection moiety is a nanoparticle, a metal nanoparticle, a noble metal nanoparticle, an alkali metal nanoparticle, a gold nanoparticle, or a silver nanoparticle having an intact stem-loop oligonucleotide attached thereto; - the second detectable signal arises from that strand of the double-stranded stem portion of the split stem-loop oligonucleotide dissociating; (i) a change in refractive index, (ii) a change in color, and / or (iii) Changes in absorption spectrum 49. The method of any one of embodiments 43 to 48, wherein

[0085] Embodiment 50. - the second detection moiety is an electrochemical agent to which an intact stem-loop oligonucleotide is attached; - The method of any one of embodiments 43 to 48, wherein the second detectable signal is a change in electrochemical signal resulting from that strand of the double-stranded stem portion of the split stem-loop oligonucleotide dissociating.

[0086] Embodiment 51. 51. The method of embodiment 50, wherein the electrochemical agent is selected from any one or more of nanoparticles, methylene blue, toluene blue, oracet blue, Hoechst 33258, [Ru(phen)3]2+, ferrocene, and / or daunomycin.

[0087] Embodiment 52. the second detection moiety is a fluorophore; The method of any one of embodiments 20 to 22, 29 to 31, 38 and 42, wherein the second detectable signal provided by said strand of the double-stranded stem portion of the split stem-loop oligonucleotide dissociating increases the distance of the fluorophore from the quencher molecule.

[0088] Embodiment 53. 53. The method of embodiment 52, wherein the fluorophore and quencher molecules are located on opposite strands of the double-stranded stem portion of the split stem-loop oligonucleotide.

[0089] Embodiment 54. the production of the first detectable signal is not reversible, - a modification to the first oligonucleotide enables the first detection moiety to provide a first detectable signal at or below a first temperature; The method of any one of embodiments 1 to 4, wherein the first detectable signal provided at or below the first temperature remains detectable at the second temperature.

[0090] Embodiment 55. -Part (c) is (i) a first background signal within 1°C, 2°C, 3°C, 4°C, or 5°C of a first temperature and a second background signal within 1°C, 2°C, 3°C, 4°C, or 5°C of a second temperature; and / or (ii) a third background signal at a third temperature; which are provided by first and second detection moieties in the mixture or in a control mixture; - Part (d) at one or more times during or after such processing: (i) determining whether a first detectable signal resulting from the modification is produced at a first temperature, the first detectable signal being distinct from a first or third background signal; at the first temperature, the second detectable signal is not different from the first or third background signals; determining that detecting a difference between the first detectable signal and the first or third background signal indicates the modification of the first oligonucleotide and the presence of the first target in the sample; (ii) determining whether a second detectable signal is produced at the second temperature that is distinct from the second or third background signal and that indicates the presence of a second target in the sample.

[0091] Embodiment 56. 56. The method of embodiment 55, wherein if the first target is present in the sample, determining whether the second detectable signal is produced at the second temperature comprises compensating for any first detectable signal present when measuring the second detectable signal.

[0092] Embodiment 57. a first signal is generated that is different from a first background signal; a second signal is generated that is different from a second background signal; the second detectable signal differs from the second background signal to a greater extent than the first detectable signal differs from the first background signal; 57. The method of embodiment 55 or embodiment 56, thereby indicating the presence of a second target in the sample.

[0093] Embodiment 58. 58. The method of embodiment 57, wherein the first temperature is less than the second temperature, the Tm of the double-stranded stem portion of the intact stem-loop oligonucleotide, and the Tm of the stem portion of the split stem-loop oligonucleotide.

[0094] Embodiment 59. 58. The method of embodiment 57, wherein the first temperature is higher than the second temperature, the Tm of the stem portion of the intact stem-loop oligonucleotide, and the Tm of the stem portion of the split stem-loop oligonucleotide.

[0095] Embodiment 60. a first signal is generated that is distinct from a third background signal; a second signal is generated that is distinct from a third background signal; the second signal differs from a third background signal to a greater extent than the first signal differs from a third background signal; 56. The method of embodiment 55, thereby indicating the presence of a second target in the sample.

[0096] Embodiment 61. the second temperature is greater than the first temperature; the third temperature is lower than the Tm of the double-stranded stem portion of the intact stem-loop oligonucleotide; a first detectable signal is produced that is distinct from a third background signal; a second detectable signal is produced that is distinct from a third background signal; the second detectable signal differs from a third background signal to a greater extent than the first signal differs from a third background signal; 56. The method of embodiment 55, thereby indicating the presence of a second target in the sample.

[0097] Embodiment 62. - the Tm of the stem portion of the intact stem-loop oligonucleotide is greater than the Tm of the stem portion of the split stem-loop oligonucleotide; - the first temperature is less than the second temperature and less than the Tm of the stem portion of the split stem-loop oligonucleotide; The method of any one of embodiments 55 to 61, wherein the second temperature is above the Tm of the stem portion of the split stem-loop oligonucleotide and below the Tm of the stem portion of the intact stem-loop oligonucleotide.

[0098] Embodiment 63. - the Tm of the stem portion of the intact stem-loop oligonucleotide is 1°C to 10°C, 1°C to 5°C, 5°C to 10°C, or more than 10°C higher than the Tm of the stem portion of the split stem-loop oligonucleotide; and / or - the first temperature is between 1°C and 10°C, between 1°C and 5°C, between 5°C and 10°C, or more than 10°C lower than the second temperature; and / or - the first temperature is between 1°C and 10°C, between 1°C and 5°C, between 5°C and 10°C, or more than 10°C lower than the Tm of the stem portion of the split stem-loop oligonucleotide; and / or - the second temperature is between 1°C and 10°C, between 1°C and 5°C, between 5°C and 10°C, or more than 10°C higher than the Tm of the stem portion of the split stem-loop oligonucleotide; and / or 63. The method of embodiment 62, wherein the second temperature is between 1°C and 10°C, between 1°C and 5°C, between 5°C and 10°C, or more than 10°C lower than the Tm of the stem portion of the intact stem-loop oligonucleotide.

[0099] Embodiment 64. 64. The method of embodiment 62 or embodiment 63, comprising measuring said third background signal, wherein the third temperature is less than the second temperature.

[0100] Embodiment 65. 65. The method of embodiment 64, wherein the third temperature is between 1°C and 10°C, between 1°C and 5°C, between 5°C and 10°C, or more than 10°C lower than the second temperature.

[0101] Embodiment 66. - the Tm of the stem portion of the intact stem-loop oligonucleotide is higher than that of the split stem-loop oligonucleotide is greater than the Tm of the stem portion of the oligonucleotide, - the first temperature is greater than the second temperature, greater than the Tm of the stem portion of the split stem-loop oligonucleotide, and greater than the Tm of the stem portion of the intact stem-loop oligonucleotide; The method of any one of embodiments 55 to 61, wherein the second temperature is above the Tm of the stem portion of the split stem-loop oligonucleotide and below the Tm of the stem portion of the intact stem-loop oligonucleotide.

[0102] Embodiment 67. - the Tm of the stem portion of the intact stem-loop oligonucleotide is 1°C to 10°C, 1°C to 5°C, 5°C to 10°C, or more than 10°C higher than the Tm of the stem portion of the split stem-loop oligonucleotide; and / or - the first temperature is between 1°C and 10°C, between 1°C and 5°C, between 5°C and 10°C, or more than 10°C higher than the second temperature; and / or - the first temperature is between 1°C and 10°C, between 1°C and 5°C, between 5°C and 10°C, or more than 10°C higher than the Tm of the stem portion of the split stem-loop oligonucleotide; and / or - the first temperature is between 1°C and 10°C, between 1°C and 5°C, between 5°C and 10°C, or more than 10°C higher than the Tm of the stem portion of the intact stem-loop oligonucleotide; and / or - the second temperature is between 1°C and 10°C, between 1°C and 5°C, between 5°C and 10°C, or more than 10°C higher than the Tm of the stem portion of the split stem-loop oligonucleotide; and / or 67. The method of embodiment 66, wherein the second temperature is between 1°C and 10°C, between 1°C and 5°C, between 5°C and 10°C, or more than 10°C lower than the Tm of the stem portion of the intact stem-loop oligonucleotide.

[0103] Embodiment 68. - the first oligonucleotide is a substrate for a multicomponent nucleic acid enzyme (MNAzyme); The mixture is further comprising an MNAzyme capable of cleaving the first oligonucleotide if the first target is present in the sample; - treating the mixture 68. The method of any one of embodiments 54-67, further comprising binding the MNAzyme to a first target and hybridizing a substrate arm of the MNAzyme to the first oligonucleotide by complementary base pairing that promotes cleavage of the first oligonucleotide, thereby providing the modification to the first oligonucleotide and enabling the first detection moiety to provide a first detectable signal.

[0104] Embodiment 69. - the first target is a nucleic acid sequence; treating the reaction mixture, 69. The method of embodiment 68, further comprising hybridizing the first target to the sensor arms of the MNAzyme by complementary base pairing, thereby promoting assembly of the MNAzyme.

[0105] Embodiment 70. - the first oligonucleotide is a substrate for an aptazyme; the first target is an analyte, a protein, a compound, or a molecule; the mixture further comprises an aptazyme comprising an aptamer capable of binding to the first target; - treating the mixture The aptazyme is bound to the first target and the first oligonucleotide to promote cleavage of the first oligonucleotide, thereby providing the modification to the first oligonucleotide. 68. The method of any one of embodiments 54-67, further comprising providing a first detection moiety and allowing the first detection moiety to generate a first detectable signal.

[0106] Embodiment 71. - the first target is a nucleic acid sequence; the first oligonucleotide comprises a sequence that is complementary to the first target; The mixture is a primer complementary to a portion of the first target, and further comprising a polymerase having exonuclease activity; - treating the mixture hybridizing a primer to a first target by complementary base pairing; hybridizing a first oligonucleotide to a first target by complementary base pairing; 68. The method of any one of embodiments 54-67, comprising extending the primer using a polymerase and the first target as a template sequence, thereby digesting the first oligonucleotide, and providing said modification to the first oligonucleotide that enables the first detection moiety to generate a first detectable signal.

[0107] Embodiment 72. - the first target is a nucleic acid sequence; The mixture is further comprising a restriction endonuclease capable of digesting a double-stranded duplex containing the first target; - treating the mixture hybridizing a first oligonucleotide to a first target by complementary base pairing, thereby forming a double-stranded duplex structure; 68. The method of any one of embodiments 54 to 67, comprising digesting the duplex structure using a restriction endonuclease, thereby providing the modification to the first oligonucleotide and enabling the first detection moiety to provide a first detectable signal.

[0108] Embodiment 73. 73. The method of embodiment 72, wherein the restriction endonuclease is a nicking endonuclease capable of associating with and cleaving a strand of the double-stranded duplex structure, said strand comprising all or a portion of the first oligonucleotide.

[0109] Embodiment 74. the mixture further comprises a DNAzyme or ribozyme that requires a cofactor for catalytic activity, - the treatment of the mixture Binding a cofactor to the DNAzyme or ribozyme to activate it catalytically; hybridizing the DNAzyme or ribozyme to the first oligonucleotide by complementary base pairing; and providing a modification to the first oligonucleotide that catalytically activates the DNAzyme or ribozyme, thereby digesting the first oligonucleotide, thereby enabling the first detection moiety to provide a first detectable signal; using conditions suitable for 68. The method of any one of embodiments 54-67, wherein the first target is a cofactor.

[0110] Embodiment 75. The cofactor is a metal ion, or Mg 2+ , Mn 2+ , Ca 2+ , Pb 2+ 75. The method of embodiment 74, wherein the metal ion is selected from:

[0111] Embodiment 76. - The method according to any one of embodiments 54 to 75, wherein the first detection moiety is a fluorophore and the modification to the first oligonucleotide increases the distance of the fluorophore from the quencher molecule.

[0112] Embodiment 77. - The method of embodiment 76, wherein the first oligonucleotide comprises a quencher molecule.

[0113] Embodiment 78. the second detection moiety is a fluorophore; 78. The method of embodiment 76 or embodiment 77, wherein the second detectable signal provided by said strand of the double-stranded stem portion of the split stem-loop oligonucleotide dissociating increases the distance of the fluorophore from the quencher molecule.

[0114] Embodiment 79. 79. The method of embodiment 78, wherein the fluorophore and quencher molecules are located on opposite strands of the double-stranded stem portion of the split stem-loop oligonucleotide.

[0115] Embodiment 80. the first detection moiety is a nanoparticle, a metal nanoparticle, a noble metal nanoparticle, an alkali metal nanoparticle, a gold nanoparticle, or a silver nanoparticle having a first oligonucleotide attached thereto; - the first detectable signal arises from the first detection moiety after said modification of the first oligonucleotide; (i) a change in refractive index, (ii) a change in color, and / or (iii) Changes in absorption spectrum 80. The method of embodiment 54 or embodiment 79, wherein

[0116] Embodiment 81. the first detection moiety is an electrochemical agent to which a first oligonucleotide is attached; - The method according to any one of embodiments 54 to 79, wherein the first detectable signal is a change in the electrochemical signal resulting from the first detection moiety after said modification of the first oligonucleotide.

[0117] Embodiment 82. 82. The method of embodiment 81, wherein the electrochemical agent is selected from any one or more of nanoparticles, methylene blue, toluene blue, oracet blue, Hoechst 33258, [Ru(phen)3]2+, ferrocene, and / or daunomycin.

[0118] Embodiment 83. the second detection moiety is a nanoparticle, a metal nanoparticle, a noble metal nanoparticle, an alkali metal nanoparticle, a gold nanoparticle, or a silver nanoparticle having an intact stem-loop oligonucleotide attached thereto; - the second detectable signal arises from that strand of the double-stranded stem portion of the split stem-loop oligonucleotide dissociating; (i) a change in refractive index, (ii) a change in color, and / or (iii) Changes in absorption spectrum 83. The method of any one of embodiments 80 to 82, wherein

[0119] Embodiment 84. - the second detection moiety is an electrochemical agent to which an intact stem-loop oligonucleotide is attached; - The method of any one of embodiments 80 to 82, wherein the second detectable signal is a change in the electrochemical signal resulting from that strand of the double-stranded stem portion of the split stem-loop oligonucleotide dissociating.

[0120] Embodiment 85. 85. The method of embodiment 84, wherein the electrochemical agent is selected from any one or more of nanoparticles, methylene blue, toluene blue, oracet blue, Hoechst 33258, [Ru(phen)3]2+, ferrocene, and / or daunomycin.

[0121] Embodiment 86. The method of any one of embodiments 1 to 85, wherein the intact stem-loop oligonucleotide is not hybridized to the second target during said digestion of one or more unhybridized nucleotides of the intact stem-loop oligonucleotide by the first enzyme.

[0122] Embodiment 87. - the first enzyme is a first MNAzyme; - treating the mixture 87. The method of any one of embodiments 1-86, comprising binding a first MNAzyme to a second target and hybridizing a substrate arm of said first MNAzyme to the loop portion of the intact stem-loop oligonucleotide, thereby digesting one or more unhybridized nucleotides of the intact stem-loop oligonucleotide and providing a split stem-loop oligonucleotide.

[0123] Embodiment 88. the second target is a nucleic acid sequence; - treating the mixture 88. The method of embodiment 87, further comprising hybridising a second target to the sensor arms of the first MNAzyme by complementary base pairing, thereby promoting assembly of the first MNAzyme.

[0124] Embodiment 89. the second target is an analyte, protein, compound or molecule; the first enzyme is an aptazyme comprising an aptamer capable of binding to a second target; - The method according to any one of embodiments 1 to 86, wherein binding of the second target to the aptamer is capable of catalytically activating the first enzyme.

[0125] Embodiment 90. 90. The method of embodiment 89, wherein the first enzyme is any one of an apta-DNAzyme, an apta-ribozyme, an apta-MNAzyme.

[0126] Embodiment 91. the second target is an analyte, protein, compound or molecule; - the first oligonucleotide is a substrate for an aptazyme; - a first enzyme comprising an aptamer portion capable of binding to a second target and an intact stem and a nucleic acid enzymatic portion capable of digesting one or more unhybridized nucleotides of the target oligonucleotide, - treating the mixture 87. The method of any one of embodiments 1 to 86, further comprising binding a second target to the aptamer portion of the aptazyme to promote activation of the catalytic activity of the nucleic acid enzyme portion, and hybridizing the intact stem-loop oligonucleotide to the active nucleic acid enzyme portion, thereby digesting one or more unhybridized nucleotides of the intact stem-loop oligonucleotide.

[0127] Embodiment 92. the second target is a nucleic acid sequence; - the first enzyme is a first restriction endonuclease, and treating the mixture includes 86. The method of any one of embodiments 1-85, comprising using conditions suitable for hybridizing a second target to the single-stranded loop portion of the intact stem-loop oligonucleotide by complementary base pairing to form a double-stranded sequence for a first restriction endonuclease to associate with and digest one or more unhybridized nucleotides of the single-stranded loop portion, thereby forming a split stem-loop oligonucleotide.

[0128] Embodiment 93. 93. The method of embodiment 92, wherein the first restriction endonuclease is a first nicking endonuclease capable of associating with and cleaving a strand of the double-stranded sequence of the first restriction endonuclease, said strand comprising all or a portion of the single-stranded loop portion of the intact stem-loop oligonucleotide.

[0129] 94. - the first enzyme comprises a polymerase with exonuclease activity, - treating the mixture hybridizing a second target to the single-stranded loop portion of the intact stem-loop oligonucleotide by complementary base pairing to form a first double-stranded sequence comprising a portion of the second target; hybridizing a first primer oligonucleotide to a second target to form a second double-stranded sequence located upstream from the first double-stranded sequence that includes a portion of the second target; using a polymerase having exonuclease activity and conditions suitable for extending the primer using the second target as a template sequence; 86. The method of any one of embodiments 1-85, wherein the first polymerase comprising exonuclease activity digests the single-stranded loop portion of the first double-stranded sequence, thereby forming a split stem-loop oligonucleotide.

[0130] 95. - the first enzyme is an exonuclease, - treating the mixture hybridizing a second target to the single-stranded loop portion of the intact stem-loop oligonucleotide by complementary base pairing to form a first double-stranded sequence comprising a portion of the second target; associating a first enzyme comprising exonuclease activity with a double-stranded sequence comprising a second target; 86. The method of any one of embodiments 1-85, comprising using conditions suitable for catalytically activating a first enzyme comprising exonuclease activity to digest a single-stranded loop portion of a first double-stranded sequence comprising a second target, thereby forming a split stem-loop oligonucleotide.

[0131] 96. - the first enzyme is a DNAzyme or a ribozyme that requires a cofactor for catalytic activity, and treating the mixture - binding a cofactor to the first enzyme to catalytically activate it; hybridizing a DNAzyme or ribozyme to the single-stranded loop portion of the intact stem-loop oligonucleotide by complementary base pairing; - using conditions suitable for catalytically activating a DNAzyme or ribozyme to digest one or more unhybridized nucleotides of the single-stranded loop portion of the intact stem-loop oligonucleotide, thereby forming a split stem-loop oligonucleotide; 86. The method of any one of embodiments 1-85, wherein the second target is a cofactor.

[0132] Embodiment 97. The cofactor is a metal ion, or Mg 2+ , Mn 2+ , Ca 2+ , Pb 2+ 97. The method of embodiment 96, wherein the metal ion is selected from:

[0133] Embodiment 98. - the first target is different from the second target, and / or - The method according to any one of embodiments 1 to 97, wherein the first oligonucleotide comprises or consists of a sequence that is not within the single-stranded loop portion of the intact stem-loop oligonucleotide.

[0134] Embodiment 99. - The method according to any one of embodiments 1 to 98, wherein the first enzyme does not digest the second target.

[0135] Embodiment 100. The method of any one of embodiments 1 to 71, 74 to 91, or 94 to 99, wherein none of the enzymes digests the first target and / or the second target.

[0136] Embodiment 101. The method of any one of embodiments 1-100, wherein the first temperature differs from the second temperature by 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 more than 60°C.

[0137] Embodiment 102. The determining step comprises: - at one or more points during the process; or The method of any one of embodiments 1-101, comprising detecting the first detectable signal and / or any said background signal at one or more time points during said treatment and at one or more time points after said treatment.

[0138] Embodiment 103. The determination is: The method according to any one of embodiments 1 to 101, comprising detecting the first detectable signal and / or any of said background signals at one or more time points after said treatment.

[0139] Embodiment 104. The determination is: - at one or more points during the process; or The method of any one of embodiments 1 to 101, comprising detecting a second detectable signal and / or any of said background signals at one or more time points during said treatment and at one or more time points after said treatment.

[0140] Embodiment 105. The determination is: The method according to any one of embodiments 1 to 101, comprising detecting a second detectable signal and / or any of said background signals at one or more time points after said treatment.

[0141] Embodiment 106. - The method according to any one of embodiments 1 to 105, wherein determining the presence or absence of said first and second targets comprises melting curve analysis.

[0142] Embodiment 107. 7. The method of embodiment 6, wherein determining the presence or absence of said first and second targets comprises a melting curve analysis comprising first and second detectable signals and optionally first and second background signals.

[0143] Embodiment 108. determining the presence or absence of the first and second targets by measuring first and second detectable signals and optionally first and second background signals, or - The method of embodiment 55, comprising a melting curve analysis comprising a first and a second detectable signal and optionally a third background signal.

[0144] Embodiment 109. The method according to any one of embodiments 1 to 108, wherein the first target and / or the second target is a nucleic acid amplicon.

[0145] Embodiment 110. - the first target is a nucleic acid and / or the second target is a nucleic acid; the mixture further comprises reagents for amplifying said first and / or second targets; 110. The method according to any one of embodiments 1 to 109, wherein treating the mixture further comprises conditions suitable for carrying out amplification of the first and / or second target.

[0146] Embodiment 111. 111. The method of embodiment 110, wherein the amplification is any one or more of polymerase chain reaction (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 (3SR), nucleic acid sequence-based amplification (NASBA), ligase chain reaction (LCR) or divergent amplification method (RAM), and / or reverse transcription polymerase chain reaction (RT-PCR).

[0147] Embodiment 112. The determination is: occurs before the amplification or within 1, 2, 3, 4, or 5 cycles of the initiation of the amplification; and / or The method of embodiment 110 or embodiment 111, which is performed after the amplification is completed.

[0148] Embodiment 113. The determination is: occurs before the amplification or within 1, 2, 3, 4, or 5 minutes of the start of the amplification; and / or The method according to any one of embodiments 110 to 112, which is carried out after the amplification is completed.

[0149] Embodiment 114. The determination is: at a first time point prior to said amplification, and The method according to any one of embodiments 110 to 113, wherein the method is performed at a second time point after completion of the amplification.

[0150] Embodiment 115. - the amplification method is polymerase chain reaction (PCR), The method according to any one of embodiments 110 to 114, wherein the determining is performed in multiple cycles, optionally in each cycle.

[0151] Embodiment 116. - normalizing a first detectable signal at a first temperature measured during or at a subsequent time point in said amplification using a positive control signal generated at a first temperature prior to said amplification and / or said treatment of the reaction mixture; and / or 112. The method of embodiment 110 or embodiment 111, further comprising normalizing a second detectable signal at a second temperature measured during or after said amplification using a positive control signal generated at a second temperature before said amplification and / or said treatment of the reactants.

[0152] Embodiment 117. - normalizing the first detectable signal using a detectable signal generated by the intact stem-loop oligonucleotide at a first temperature prior to said amplification and / or said treatment of the reaction mixture; and / or - normalizing a second detectable signal using a detectable signal generated by an intact stem-loop oligonucleotide at an additional temperature prior to said amplification and / or said treatment of the reaction; 112. The method of embodiment 110 or embodiment 111, wherein the additional temperature is above the Tm of the intact stem-loop oligonucleotide.

[0153] Embodiment 118. The method according to any one of embodiments 1 to 117, further comprising generating a first target positive control signal using a known concentration of the first target and / or a known concentration of the modified first oligonucleotide.

[0154] Embodiment 119. 119. The method of any one of embodiments 1 to 118, further comprising generating a first target positive control signal by repeating the method on a separate control sample containing said first target.

[0155] Embodiment 120. 120. The method of embodiment 119, wherein the separate control sample comprising the first target comprises a known concentration of the first target.

[0156] Embodiment 121. The method of embodiment 119 or embodiment 120, wherein the separate control sample comprising the first target further comprises a second target.

[0157] Embodiment 122. - a known concentration of a second target and / or a known concentration of the modified stem-loop oligonucleotide 122. The method of any one of embodiments 1-121, further comprising generating a second target positive control signal using a nucleotide.

[0158] Embodiment 123. 123. The method of any one of embodiments 1 to 122, further comprising generating a second target positive control signal by repeating the method on a separate control sample containing a second target.

[0159] Embodiment 124. 124. The method of embodiment 123, wherein the control sample comprising the second target comprises a known concentration of the second target.

[0160] Embodiment 125. The method of embodiment 123 or embodiment 124, wherein said control sample containing a second target further comprises said first target.

[0161] Embodiment 126. - The method of any one of embodiments 1 to 125, further comprising generating a combined positive control signal by repeating the method on separate control samples comprising the first target and the second target.

[0162] Embodiment 127. 127. The method of embodiment 126, wherein the combined control sample comprises a known concentration of the first target and / or a known concentration of the second target.

[0163] Embodiment 128. The method according to any one of embodiments 116 to 127, further comprising normalizing the first detectable signal and / or the second detectable signal using any said positive control signal.

[0164] Embodiment 129. (i) the first target; or (ii) the second target; or (iii) assessing the level of the negative control signal by repeating the method of any one of embodiments 1 to 115 on a separate negative control sample that does not contain the first target or the second target.

[0165] Embodiment 130. 130. The method of embodiment 129, further comprising normalizing the first detectable signal and / or the second detectable signal using said negative control signal.

[0166] Embodiment 131. - The method according to any one of embodiments 116 to 130, wherein any said control signal is a fluorescent control signal.

[0167] Embodiment 132. The method further comprising comparing the first and / or second detectable signal to a threshold value; - the threshold value is generated using a detectable signal derived from a series of samples or derivatives thereof tested according to the method according to any one of embodiments 1 to 115, (i) No-template control and first target (ii) No-template control and second target (iii) No-template control, primary target, and secondary target 132. The method of any one of embodiments 1 to 131, comprising any one or more of:

[0168] Embodiment 133. The method of embodiment 132, wherein a series of samples or derivatives thereof are tested using a known concentration of the first oligonucleotide and / or a known concentration of the intact stem-loop oligonucleotide.

[0169] Embodiment 134. The method according to any one of embodiments 1 to 133, wherein the sample is a biological sample obtained from a subject.

[0170] Embodiment 135. - The method according to any one of embodiments 1 to 133, wherein the method is carried out in vitro.

[0171] Embodiment 136. - The method according to any one of embodiments 1 to 133, wherein the method is carried out ex vivo.

[0172] Embodiment 137. The method according to any one of embodiments 1 to 136, wherein the first and second detectable moieties emit in the same color region of the visible spectrum.

[0173] Embodiment 138. a first oligonucleotide for detecting a first target, the first target being a nucleic acid and complementary to at least a portion of the first oligonucleotide; a first detection moiety, the first detection moiety is capable of generating a first detectable signal upon modification of the first oligonucleotide; a first detection moiety, wherein the modification is induced by hybridization to a first oligonucleotide of a first target by complementary base pairing; an intact stem-loop oligonucleotide for the detection of a second target, the intact stem-loop oligonucleotide comprising a double-stranded stem portion of hybridized nucleotides, the opposite strand of which is connected by an unbroken single-stranded loop portion of unhybridized nucleotides, at least one strand of the double-stranded stem portion comprising a second detection moiety; and a first enzyme capable of digesting one or more of the unhybridized nucleotides of the intact stem-loop oligonucleotide only if the second target is present in the sample, thereby disrupting the single-stranded loop portion and providing a split stem-loop oligonucleotide; and A composition comprising: - the second detection moiety is capable of producing a second detectable signal upon dissociation of the double-stranded stem portion of the split stem-loop oligonucleotide; A composition wherein the first and second detection moieties are capable of producing indistinguishable detectable signals at a single temperature using a single type of detector.

[0174] Embodiment 139. - The composition of embodiment 138, wherein the region of the first oligonucleotide that is complementary to the first target has a different melting temperature (Tm) for each strand of the double-stranded stem portion of the intact stem-loop oligonucleotide.

[0175] Embodiment 140. The first oligonucleotide comprises: each strand of the double-stranded stem portion of the intact stem-loop oligonucleotide, and The composition of embodiment 138 or embodiment 139, which differs in sequence from the single-stranded loop portion of the intact stem-loop oligonucleotide.

[0176] Embodiment 141. The composition of any one of embodiments 138 to 140, wherein the first oligonucleotide is a stem-loop oligonucleotide comprising a double-stranded stem portion of hybridized nucleotides on opposite strands connected by an unbroken single-stranded loop portion of unhybridized nucleotides, all or part of which is complementary to the first target.

[0177] Embodiment 142. 142. The composition of embodiment 141, wherein the first target hybridizes to the first oligonucleotide by complementary base pairing, causing strand dissociation in the double-stranded stem portion of the first oligonucleotide, thereby enabling the first detection moiety to provide a first detectable signal.

[0178] Embodiment 143. the first oligonucleotide is a double-stranded stem portion of hybridized nucleotides, the opposing strand of which is connected by a single-stranded loop portion of unhybridized nucleotides, all or part of which is complementary to the first target; a second single-stranded portion extending in the 3' direction from one of the opposing strands and terminating in a sequence complementary to a portion of the first target; 141. The composition of any one of embodiments 138-140, wherein the stem-loop oligonucleotide comprises a blocker molecule preceding a sequence complementary to a portion of the first target.

[0179] Embodiment 144. - the first target hybridizes to its second single-stranded portion by complementary base pairing; the composition further comprises a polymerase capable of extending a second single-stranded portion using the first target as a template sequence to provide a double-stranded nucleic acid, and the blocker molecule is capable of preventing the polymerase from extending the first target using the one opposing strand as a template; 144. The composition of embodiment 143, wherein upon denaturing the double-stranded nucleic acid, the second single-stranded portion extended by the polymerase hybridizes to the single-stranded loop portion of the first oligonucleotide by complementary base pairing to generate a signaling duplex structure, thereby enabling the first detection moiety to provide a first detectable signal.

[0180] Embodiment 145. - The composition of any one of embodiments 141 to 144, wherein the first detection moiety is a fluorophore.

[0181] Embodiment 146. - The composition of embodiment 145, wherein the first oligonucleotide comprises a quencher molecule, and the fluorophore and quencher molecule are located on opposite strands of the double-stranded stem portion of the first oligonucleotide.

[0182] Embodiment 147. the first oligonucleotide is a first double-stranded portion of hybridized nucleotides, the first strand extending to a single-stranded portion terminating in a complementary sequence capable of hybridizing to a portion of a first target, the first strand including a blocker molecule preceding the complementary sequence; - The composition according to any one of embodiments 138 to 140, wherein the composition further comprises a polymerase.

[0183] Embodiment 148. a portion of the first target hybridizes to the complementary sequence of the single-stranded portion by complementary base pairing; the composition further comprises a polymerase capable of extending the complementary sequence using the first target as a template sequence to provide a second double-stranded portion, and the blocker molecule prevents the polymerase from using the single-stranded portion as a template to extend the first target; 148. The composition of embodiment 147, wherein upon denaturation of the first and second double-stranded portions, the complementary sequence extended by the polymerase can hybridize to the first strand of the first double-stranded portion by complementary base pairing to generate a signaling duplex structure, thereby enabling the first detection moiety to provide a first detectable signal.

[0184] Embodiment 149. The composition of embodiment 147 or embodiment 148, wherein the first detection moiety is a fluorophore and the modification increases its distance from the quencher molecule.

[0185] 150. - The composition of embodiment 149, wherein the first oligonucleotide comprises a quencher molecule, and the fluorophore and quencher molecule are located on opposite strands of the first double-stranded portion.

[0186] Embodiment 151. - the first oligonucleotide is complementary to a first portion of the target; the composition further comprises an additional oligonucleotide complementary to a second portion of the first target, wherein the first and second portions of the first target are adjacent to but do not overlap each other, and each hybridizes to the first target; (i) a first double-stranded component by hybridizing a first oligonucleotide to a target or by complementary base pairing; and (ii) forming a duplex structure comprising a second double-stranded component by hybridizing an additional oligonucleotide to the target by complementary base pairing; The composition of any one of embodiments 138 to 140, thereby bringing the first and additional oligonucleotides into close proximity and enabling the first detection moiety to provide a first detectable signal.

[0187] Embodiment 152. - the first detectable moiety is a fluorophore and the additional oligonucleotide comprises a quencher; forming the bilayer structure further brings the fluorophore and quencher into close proximity; - the detectable signal is a decrease in fluorescence provided by the first detection moiety; The composition of embodiment 151.

[0188] Embodiment 153. a first oligonucleotide hybridizes to a first target by complementary base pairing; the composition comprising: a primer hybridized to a portion of the first target by complementary base pairing; and 141. The method of any one of embodiments 138-140, further comprising a polymerase with exonuclease activity capable of extending the primer using the first target as a template sequence, thereby digesting the first oligonucleotide and modifying the first oligonucleotide to enable the first detection moiety to provide a first detectable signal.

[0189] Embodiment 154. - the first target hybridizes to the first oligonucleotide by complementary base pairing, thereby forming a double-stranded duplex structure; The composition of any one of embodiments 138 to 140, wherein the composition further comprises a restriction endonuclease capable of digesting a double-stranded duplex structure comprising the first target, thereby modifying the first oligonucleotide and enabling the first detection moiety to provide a first detectable signal.

[0190] Embodiment 155. The composition of embodiment 154, wherein the restriction endonuclease is a nicking endonuclease capable of associating with and cleaving a strand of the double-stranded duplex structure, said strand comprising the first oligonucleotide.

[0191] Embodiment 156. - The composition according to any one of embodiments 153 to 155, wherein the first detection moiety is a fluorophore and said modification of the first oligonucleotide increases the distance of the fluorophore from the quencher molecule.

[0192] Embodiment 157. - The composition of embodiment 156, wherein the first oligonucleotide comprises a quencher molecule.

[0193] Embodiment 158. a first oligonucleotide for the detection of a first target comprising a first detection moiety, the first detection moiety is capable of generating a first detectable signal upon modification of the first oligonucleotide; a first oligonucleotide, the modification of which is induced by a first target; an intact stem-loop oligonucleotide for the detection of a second target, the intact stem-loop oligonucleotide comprising a double-stranded stem portion of hybridized nucleotides, the opposite strand of which is connected by an unbroken single-stranded loop portion of unhybridized nucleotides, at least one strand of the double-stranded stem portion comprising a second detection moiety; and a first enzyme capable of digesting one or more of the unhybridized nucleotides of the intact stem-loop oligonucleotide only if the second target is present in the sample, thereby disrupting the single-stranded loop portion and providing a split stem-loop oligonucleotide; and A composition comprising: - the second detection moiety is capable of producing a second detectable signal upon dissociation of the double-stranded stem portion of the split stem-loop oligonucleotide; A composition wherein the first and second detection moieties are capable of producing indistinguishable detectable signals at a single temperature using a single type of detector.

[0194] Embodiment 159. The first oligonucleotide comprises: each strand of the double-stranded stem portion of the intact stem-loop oligonucleotide, and The composition of embodiment 158, which differs in sequence from the single-stranded loop portion of the intact stem-loop oligonucleotide.

[0195] Embodiment 160. - the first target is a nucleic acid sequence; the composition comprising: a first primer complementary to a first sequence in a first target; a second oligonucleotide comprising a component complementary to a second sequence in the first target that is different from the first sequence, and a tag portion that is not complementary to the first target; a first polymerase comprising an exonuclease activity; 160. The composition of embodiment 158 ​​or embodiment 159, optionally further comprising a second polymerase.

[0196] Embodiment 161. a first primer and a second oligonucleotide, each hybridized to a first target by complementary base pairing; the first polymerase may extend the first primer using the target as a template, thereby cleaving the tag portion and allowing the cleaved tag portion to hybridize to the first oligonucleotide by complementary base pairing; 161. The composition of embodiment 160, wherein the first polymerase or the optional second polymerase can extend the tag portion using the first oligonucleotide as a template to generate a double-stranded sequence comprising the first oligonucleotide, thereby modifying the first oligonucleotide and enabling the first detection moiety to provide a first detectable signal.

[0197] Embodiment 162. The composition of embodiment 160 or embodiment 161, wherein the first oligonucleotide comprises a fluorophore and a quencher molecule.

[0198] Embodiment 163. the first oligonucleotide comprises a fluorophore and a quencher molecule; The composition of embodiment 162, wherein extending the tag portion increases the distance between the fluorophore and the quencher molecule.

[0199] Embodiment 164. - the first target is a cofactor for the catalytic activity of an enzyme; the composition further comprises a DNAzyme or ribozyme that requires a cofactor for catalytic activity; The composition of embodiment 158 ​​or embodiment 159, wherein the DNAzyme or ribozyme binds to a first target and hybridizes to the first oligonucleotide through complementary base pairing, thereby digesting and modifying the first oligonucleotide, and is capable of causing the first detection moiety to generate a first detectable signal.

[0200] Embodiment 165. The cofactor is a metal ion, or Mg 2+ , Mn 2+ , Ca 2+ , Pb 2+ 165. The composition of embodiment 164, wherein the metal ion is selected from:

[0201] Embodiment 166. - the first oligonucleotide is a substrate for a multicomponent nucleic acid enzyme (MNAzyme); - the composition further comprises an MNAzyme capable of cleaving the first oligonucleotide if the first target is present in the sample; 159. The method of embodiment 158 ​​or embodiment 159, wherein the MNAzyme binds to a first target and is capable of hybridizing to a first oligonucleotide by complementary base pairing via its substrate arm, said hybridization promoting cleavage of the first oligonucleotide, thereby modifying it and enabling the first detection moiety to provide a first detectable signal.

[0202] Embodiment 167. - the first target is a nucleic acid sequence; The composition of embodiment 166, wherein the first target hybridizes to the sensor arms of the MNAzyme by complementary base pairing, thereby promoting assembly of the MNAzyme.

[0203] Embodiment 168. the first target is an analyte, a protein, a compound, or a molecule; - the first oligonucleotide is a substrate for an aptazyme; The composition of embodiment 158 ​​or embodiment 159, wherein the composition further comprises an aptazyme comprising an aptamer portion capable of binding to the first target and a nucleic acid enzymatic portion capable of digesting the first oligonucleotide, thereby modifying it to enable the first detection portion to provide a first detectable signal.

[0204] Embodiment 169. The composition of embodiment 168, wherein the first target binds to the aptamer portion of the aptazyme, and the first oligonucleotide hybridizes to the active nucleic acid enzyme portion by complementary base pairing to promote digestion of the first oligonucleotide, thereby modifying it and enabling the first detection portion to provide a first detectable signal.

[0205] Embodiment 170. - The composition of any one of embodiments 166 to 169, wherein the first detection moiety is a fluorophore and modifying said first oligonucleotide increases the distance of the fluorophore from the quencher molecule.

[0206] Embodiment 171. - The composition of embodiment 170, wherein the first oligonucleotide comprises a quencher molecule.

[0207] Embodiment 172. the first detection moiety is a nanoparticle, a metal nanoparticle, a noble metal nanoparticle, an alkali metal nanoparticle, a gold nanoparticle, or a silver nanoparticle having a first oligonucleotide attached thereto; - the first detectable signal arises from the first detection moiety after said modification of the first oligonucleotide; (i) a change in refractive index, (ii) a change in color, and / or (iii) Changes in absorption spectrum 169. The composition of any one of embodiments 138-144, 147, 148, 151, 153-155, 158-161, and 164-169, wherein

[0208] Embodiment 173. the first detection moiety is an electrochemical agent to which a first oligonucleotide is attached; The composition of embodiment 172, wherein the first detectable signal is a change in an electrochemical signal.

[0209] Embodiment 174. The composition of embodiment 173, wherein the electrochemical agent is selected from any one or more of nanoparticles, methylene blue, toluene blue, oracet blue, Hoechst 33258, [Ru(phen)3]2+, ferrocene, and / or daunomycin.

[0210] Embodiment 175. the second detection moiety is a nanoparticle, a metal nanoparticle, a noble metal nanoparticle, an alkali metal nanoparticle, a gold nanoparticle, or a silver nanoparticle to which at least one strand of the double-stranded stem portion of the second oligonucleotide is attached; a second detectable signal is generated upon said dissociation of the double-stranded stem portion of the split stem-loop oligonucleotide; (i) a change in refractive index, (ii) a change in color, and / or (iii) Changes in absorption spectrum 175. The composition of any one of embodiments 172 to 174, wherein

[0211] Embodiment 176. the second detection moiety is an electrochemical agent to which a second oligonucleotide is attached; - The composition of any one of embodiments 172 to 174, wherein the second detectable signal is a change in an electrochemical signal that occurs upon said dissociation of the double-stranded stem portion of the split stem-loop oligonucleotide.

[0212] Embodiment 177. The composition of embodiment 176, wherein the electrochemical agent is selected from any one or more of nanoparticles, methylene blue, toluene blue, oracet blue, Hoechst 33258, [Ru(phen)3]2+, ferrocene, and / or daunomycin.

[0213] Embodiment 178. the second detection moiety is a fluorophore; 172. The composition of any one of embodiments 145, 146, 149, 150, 152, 156, 157, 162, 163, 170, and 171, wherein the second detectable signal provided by said second detection moiety upon dissociation of the double-stranded stem portion of the split stem-loop oligonucleotide increases the distance of the fluorophore from the quencher molecule.

[0214] Embodiment 179. The composition of embodiment 178, wherein the fluorophore and quencher molecules are located on opposite strands of the double-stranded stem portion of the stem-loop oligonucleotide.

[0215] Embodiment 180. - the first enzyme is a first MNAzyme; - the first MNAzyme binds to a second target; - the substrate arm of said first MNAzyme hybridizes to the single loop portion of the intact stem-loop oligonucleotide by complementary base pairing, thereby forming an intact stem-loop oligonucleotide. 179. The composition of any one of embodiments 138 to 179, wherein the composition promotes digestion of one or more unhybridized nucleotides of a oligonucleotide to provide a split stem-loop oligonucleotide.

[0216] Embodiment 181. the second target is a nucleic acid sequence, The composition of embodiment 180, wherein the second target hybridizes to the sensor arms of the first MNAzyme by complementary base pairing, thereby promoting assembly of the first MNAzyme.

[0217] Embodiment 182. the second target is an analyte, protein, compound or molecule; the first enzyme is an aptazyme comprising an aptamer capable of binding to a second target; - The composition of any one of embodiments 138 to 179, wherein the aptamer binds to a second target, thereby catalytically activating the first enzyme.

[0218] Embodiment 183. The composition of embodiment 182, wherein the first enzyme is any one of an apta-DNAzyme, an apta-ribozyme, an apta-MNAzyme.

[0219] Embodiment 184. the second target is an analyte, protein, compound or molecule; - the single-stranded loop portion of the intact stem-loop oligonucleotide is a substrate for an aptazyme; The composition of any one of embodiments 138 to 179, wherein the composition further comprises an aptazyme comprising an aptamer portion capable of binding to a second target and a nucleic acid enzymatic portion capable of digesting one or more unhybridized nucleotides of the intact stem-loop oligonucleotide, thereby forming a split stem-loop oligonucleotide.

[0220] Embodiment 185. The composition of embodiment 184, wherein the second target binds to the aptamer portion of the aptazyme and the single-stranded loop portion of the intact stem-loop oligonucleotide hybridizes to the active nucleic acid enzyme portion by complementary base pairing to promote digestion of one or more unhybridized nucleotides of the intact stem-loop oligonucleotide, thereby forming a split stem-loop oligonucleotide.

[0221] Embodiment 186. the second target is a nucleic acid sequence; - the first enzyme is a first restriction endonuclease, 179. The composition of any one of embodiments 138 to 179, wherein the second target is hybridized to the single-stranded loop portion of the intact stem-loop oligonucleotide by complementary base pairing, and the first restriction endonuclease associates with and digests one or more unhybridized nucleotides of the intact stem-loop oligonucleotide, forming a double-stranded sequence for digestion thereof, thereby forming the split stem-loop oligonucleotide.

[0222] Embodiment 187. 187. The composition of embodiment 186, wherein the first restriction endonuclease is a first nicking endonuclease capable of associating with and cleaving a strand of the double-stranded sequence of the first restriction endonuclease, wherein the strand comprises an intact stem-loop oligonucleotide.

[0223] Embodiment 188. - the first enzyme comprises a polymerase with exonuclease activity, - a second target is hybridized to the single-stranded loop portion of the intact stem-loop oligonucleotide by complementary base pairing to form a first double-stranded sequence that includes a portion of the second target; the composition further comprises a first primer oligonucleotide that hybridizes to the second target by complementary base pairing to form a second double-stranded sequence located upstream relative to the first double-stranded sequence that comprises a portion of the second target; The composition of any one of embodiments 138 to 179, wherein the primer can be extended using a polymerase having exonuclease activity and the second target as a template sequence to digest the single-stranded loop portion of the first double-stranded sequence, thereby forming a split stem-loop oligonucleotide.

[0224] Embodiment 189. - the first enzyme is an exonuclease, 179. The composition of any one of embodiments 138 to 179, wherein the second target is hybridized to the single-stranded loop portion of the intact stem-loop oligonucleotide by complementary base pairing to form a first double-stranded sequence comprising a portion of the second target, to which a first enzyme comprising exonuclease activity can associate, thereby digesting the single-stranded loop portion of the first double-stranded sequence comprising the second target to form a split stem-loop oligonucleotide.

[0225] Embodiment 190. - the first enzyme is a DNAzyme or ribozyme that requires a cofactor for catalytic activity, the second target is a cofactor and is bound to the DNAzyme or ribozyme; 179. The composition of any one of embodiments 138 to 179, wherein the DNAzyme or ribozyme hybridizes to the single-stranded loop portion of the intact stem-loop oligonucleotide by complementary base pairing and digests one or more unhybridized nucleotides of the single-stranded loop portion of the intact stem-loop oligonucleotide, thereby forming a split stem-loop oligonucleotide.

[0226] Embodiment 191. The cofactor is a metal ion, or Mg 2+ , Mn 2+ , Ca 2+ , Pb 2+ 191. The composition of embodiment 190, wherein the metal ion is selected from:

[0227] Embodiment 192. - The composition of any one of embodiments 138-150, 153, 156-158, 166 or 167, wherein the first oligonucleotide is selected from any one or more of a Molecular Beacon®, a Scorpions® primer, a TaqMan® primer, or an MNAzyme substrate.

[0228] Embodiment 193. - The composition according to any one of embodiments 138 to 192, wherein the first target and / or the second target is a nucleic acid amplicon.

[0229] Embodiment 194. A method for determining the presence or absence of a first and a second target in a sample, comprising: (a) a sample or derivative thereof suspected of containing first and second targets or amplicons thereof, a first oligonucleotide for the detection of a first target or an amplicon thereof, the first oligonucleotide comprising a first detection moiety capable of generating a first detectable signal; an intact stem-loop oligonucleotide for the detection of a second target or an amplicon thereof, comprising a double-stranded stem portion of hybridized nucleotides, the opposite strand of which is connected by an unbroken single-stranded loop portion of unhybridized nucleotides, the stem portion comprising a second detection moiety capable of generating a second detectable signal; The first and second detectable signals are indistinguishable at a single temperature using a single type of detector, an intact stem-loop oligonucleotide, and - preparing a reaction mixture by contacting with a first enzyme capable of digesting one or more unhybridized nucleotides of the intact stem-loop oligonucleotide only if a second target or an amplicon thereof is present in the sample; (b) adding the mixture to - the first target or an amplicon thereof is suitable for inducing a modification to the first oligonucleotide, thereby enabling the first detection moiety to provide a first detectable signal; - treating under conditions suitable for digesting one or more of the unhybridized nucleotides of the intact stem-loop oligonucleotide with a first enzyme only if the second target or an amplicon thereof is present in the sample, thereby disrupting the single-stranded loop portion and providing a split stem-loop oligonucleotide; (c) a first background signal within 5°C of a first temperature and a second background signal within 5°C of a second temperature; or - a third background signal at a third temperature and measuring the first and second detection moieties provided in the mixture or in the control mixture; (d) at one or more points during or after such processing: determining whether a first detectable signal distinct from a first or third background signal is produced at a first temperature; the second detectable signal produced at the first temperature is not different from the first or third background signals; determining that detecting a difference between the first detectable signal and the first or third background signal indicates the modification of the first oligonucleotide and the presence of the first target or an amplicon thereof in the sample; - determining whether a second detectable signal distinct from a second or third background signal is produced at the second temperature; the strands of the double-stranded stem portion of the split stem-loop oligonucleotide dissociate, allowing the second detection moiety to provide a second detectable signal indicative of the presence of a second target or an amplicon thereof in the sample; determining that the strands of the double-stranded stem portion of the intact stem-loop oligonucleotide are unable to dissociate, thereby ensuring inhibition of the second detectable moiety and the absence of a second detectable signal indicating the absence of the second target in the sample.

[0230] Preferred embodiments of the present invention will now be described, by way of example only, with reference to the accompanying figures identified below. [Brief explanation of the drawings]

[0231] [Figure 1]Exemplary LOCS reporters and their melting temperatures (Tm) are shown in intact and split conformations. The illustrated LOCS reporters can be used in combination with various standard reporter probes and substrates known in the art for nucleic acid detection. An exemplary intact LOCS reporter (Figure 1A, LHS, top and bottom) has a loop region, a stem region, and a detection moiety, such as a fluorophore (F)-quencher (Q) dye pair, that can be cleaved or degraded. Cleavage or degradation of the loop region in the presence of target can generate a split LOCS reporter structure (Figure 1B, RHS, top and bottom). The melting temperature of the stem region of the intact LOCS (Tm A) is higher than the Tm of the stem region in the split LOCS (Tm B). Therefore, the stem of the intact LOCS melts and separates at temperatures above Tm A. In contrast, the stem of the split LOCS, which retains the two fragments, melts and separates at temperatures above Tm B, resulting in increased fluorescence. [Figure 2] An exemplary strategy for target detection using LOCS oligonucleotides, which are universal and can be used to detect any target, is shown. In this scheme, the LOCS oligonucleotide contains a stem region, a fluorophore-quencher dye pair, and a loop region. The loop region contains a universal substrate for a catalytic nucleic acid, such as an MNAzyme, also known in the art as a PlexZyme. MNAzymes are formed when the target sensor arms of the constituent partzymes align adjacently on a target. The loop region of the LOCS oligonucleotide binds to the substrate-binding arm of the assembled MNAzyme, and the substrate in the LOCS loop is cleaved by the MNAzyme, generating a split-LOCS structure. Both intact and split LOCS are quenched or generate fluorescence depending on whether the temperature of the reaction environment is above or below the melting temperatures of their stems, i.e., Tm A and Tm B, respectively. The presence of fluorescence at temperatures between Tm B and Tm A indicates the presence of a target that promotes cleavage. The target may be detected directly, or the target amplicon generated by a target amplification protocol may be detected. [Figure 3]In this example, a linear MNAzyme substrate is used in combination with a single LOCS probe containing an MNAzyme substrate within its loop. Both the linear MNAzyme substrate and the single LOCS probe are labeled with the same detection moiety, e.g., a specific fluorophore (F) / quencher (Q) dye pair. The linear substrate contains a first substrate sequence cleavable by a first MNAzyme that assembles in the presence of a first target 1 (FIG. 3A). In the presence of target 1, the linear substrate is cleaved, resulting in an increase in fluorescence that can be detected at all temperatures. The LOCS probe contains a second substrate sequence cleavable by a second MNAzyme that assembles in the presence of a second target 2 (FIG. 3B). In the presence of target 2, the LOCS substrate is cleaved, generating a split LOCS that melts at a melting temperature (Tm B) lower than the melting temperature (Tm A) of the intact LOCS. At temperatures below Tm B, the stem portions of the split LOCS remain hybridized (closed) and therefore quenched. At temperatures above Tm B, the stem portions of the split LOCS dissociate (separate), resulting in an increase in fluorescence. If both targets are present and fluorescence is measured at a temperature below Tm B, the increase in fluorescence can be associated with target 1 only; if fluorescence is measured at a temperature above Tm B but below Tm A, the increase in fluorescence can be associated with target 1 and / or target 2. [Figure 4]An exemplary strategy for target detection using target-specific LOCS oligonucleotides is shown, which can be used in combination with other types of reporter probes or substrates, such as standard TaqMan, molecular beacons, Scorpion uniprobes, Scorpion biprobes, or linear MNAzyme substrate probes. An intact LOCS oligonucleotide can contain a stem region, a fluorophore-quencher dye pair, and a loop region that contains a region complementary to the target amplicon. In the scheme shown in Figure 4A, the loop region of the LOCS oligonucleotide is complementary to the target amplicon and binds to it during amplification. During primer extension, the exonuclease activity of the polymerase degrades the loop region but leaves the stem region intact. The stem regions are complementary to each other but not to their targets. Degradation generates split LOCS oligonucleotides, where the stems remain hybridized and are quenched at temperatures below the stem's Tm. Raising the temperature above the stem's Tm causes the strands to separate and fluorescence can be emitted. In the scheme shown in Figure 4B, the loop region of the LOCS oligonucleotide contains a region complementary to the target amplicon and further contains a recognition site for a restriction enzyme, e.g., a nicking enzyme. The loop region of the LOCS oligonucleotide binds to the target, and the nicking enzyme cleaves the loop region, leaving the target molecule intact. This splits the LOCS, and a fluorescent signal is emitted at temperatures above the melting temperature of the stem. At lower temperatures, the stem regions of the split LOCS structure can anneal and quench the fluorescence. This strategy can be used to detect target sequences directly or to detect target amplicons when combined with target amplification methods. [Figure 5]

[0033] Figure 1 illustrates an embodiment in which a non-cleavable molecular beacon can be combined with a LOCS probe that is cleavable by an MNAzyme. Both the molecular beacon and the LOCS probe can be labeled with the same fluorophore. The molecular beacon can have a stem region with Tm A and a loop region capable of specifically hybridizing to a first target 1 with Tm B, where Tm B is higher than Tm A. This can be combined with an intact LOCS probe that can have a stem region with Tm C and a loop region that can be cleaved by an MNAzyme in the presence of a second target 2, thus generating a split LOCS probe with Tm D, where Tm D is less than Tm C. The presence of target 1 and / or target 2 can be identified by measuring fluorescence at two temperatures in real time, or by using separate measurements taken at or near the start of amplification and after amplification. [Figure 6] Figure 6 shows exemplary PCR amplification curves for quantitative analysis of a first target 1 (CTcry) at a first temperature in the presence or absence of various concentrations of a second target 2 (NGopa). The protocol combined one linear MNAzyme substrate (for target 1) with one LOCS reporter (for target 2). Results were obtained in the HEX channel for quantitative detection of CTcry (target 1) at an acquisition temperature of 52 °C for reactions containing 20,000 (black dots), 4,000 (black dashed line), 800 (black squares), 160 (gray solid line), or 32 (gray dots) copies of the CTcry template alone (Figure 6A), or in a background of 20,000 (Figure 6B) or 32 (Figure 6C) copies of NGopa (target 2). Fluorescence data were also collected at 52 °C for reactions lacking CTcry but containing either 20,000 (black line) or 32 copies (gray line) of NGopa template (Figure 6D). A no-target control (nuclease-free HO) is shown in Figures 6A-6C (solid black line). Amplification curves are the average of fluorescence levels from triplicate reactions. [Figure 7]Figure 7A shows the simultaneous qualitative detection of target 1 (CTcry) and / or target 2 (NGopa) at two temperatures (D1 and D2) using endpoint assay method 1 in the HEX channel. The presented results are the average of triplicate reactions, and error bars represent the standard deviation between these replicates. Specifically, the data show the change in fluorescence signal of one linear substrate and one LOCS probe, allowing for the detection and discrimination of CTcry (CT copy numbers: 20K, 4, 800, 160, 32, or 0) and / or NGopa (NG copy numbers: 20K, 32, or 0) targets, respectively, present in a background of human genomic DNA. Additionally, the presence of the human TFRC gene in genomic DNA was measured in the Texas Red channel for use as a calibrator. Figure 7A shows the signal change (ΔS, where ΔS = SD - post-PCR - SD - pre-PCR). ΔS at temperature 1 (ΔSD1, 52°C) is shown as a black and white pattern, and ΔS at temperature 2 (ΔSD2, 70°C) is shown in gray. The results show that the signal at Temperature 1 (ΔSD1, black bar) exceeds Threshold 1 (X1) when CTcry is present in the sample, but does not exceed this threshold when only NGopa is present. Therefore, a ΔSD1 greater than Threshold 1 at Temperature 1 indicates the presence of the first target (CTcry). The results in Figure 7A also indicate that NGopa is present in the sample when the signal change at Temperature 2 (ΔSD2, gray bar) is greater than the change at Temperature 1 (ΔSD2 > ΔSD1) and greater than Threshold X1 (ΔSD2 > X1). The results in Figures 7B and 7C demonstrate the use of calibrator signals to calibrate ΔSD1 and ΔSD2. Figure 7B shows the change in the TFRC calibrator signal (ΔC) measured in the Texas Red channel; values ​​above threshold C indicate a positive signal for ΔC, and values ​​below threshold C indicate a negative signal for ΔC(NTC). A calibrator template (TFRC) was present in human genomic DNA in the background for all samples except the no-target control (NF HO). Figure 7C shows the change in signal at each temperature (ΔS / ΔC) calibrated to ΔC.The signal change at temperature 1 (ΔSD1 / ΔC, 52°C) is shown as a black and white pattern, and the signal change at temperature 2 (ΔSD2 / ΔC, 70°C) is shown in gray; results were obtained for the positive reactions at ΔC in Figure 7B, but not for the negative reactions at ΔC (shown as not applicable (N / A)). Furthermore, the data in Figure 7C indicate that signal calibration does not alter the results obtained using endpoint assay 1 (Figure 7A), as the patterns are consistent. [Figure 8] Figure 1 shows the simultaneous qualitative detection of a first target (CTcry) and / or a second target (NGopa) at two temperatures using analytical method 2. CTcry (CT copy numbers 20K, 4, 800, 160, 32, or 0) and / or NGopa (NG copy numbers 20K, 32, or 0) targets were present in a background of human genomic DNA. The data show the difference in the fluorescence signal change (ΔSD2 minus ΔSD1 = ΔΔSD2ΔSD1) obtained in the HEX channel at temperature 2 (D2, 70°C) and temperature 1 (D1, 52°C) using analytical method 2. The results show that ΔΔSD2ΔSD1 exceeds threshold 2 (X2) when NGopa is present in the sample, but does not exceed this threshold when only CTcry is present and / or when NGopa is absent from the sample (NTC). Therefore, a difference in the fluorescence signal change above threshold 2 (ΔΔSD2ΔSD1 > X2) indicates the presence of NGopa. [Figure 9]Figure 9 shows the change in fluorescence signal (ΔS) obtained during PCR in the HEX channel at two different temperatures (52°C and 70°C) in the presence of a first target (CTcry) and / or a second target (NGopa) using endpoint analysis method 3. CTcry (CT copy numbers 20K, 4, 800, 160, 32, or 0) and / or NGopa (NG copy numbers 20K, 32, or 0) targets were present in a background of human genomic DNA. Figure 9A shows ΔS (ΔSD2) at temperature 2. If ΔSD2 is greater than threshold X1, this indicates that CTcry and / or NGopa are present in the sample. If ΔSD2 is less than threshold X1, this indicates that neither CTcry nor NGopa are present in the reaction. Figure 9B shows the ratio ΔSD1:ΔSD2, which is used to indicate which targets are present in the reaction. If this ratio is greater than threshold R1, this indicates that CTcry is present but NGopa is not. If the ratio is below the threshold R2, this indicates that NGopa is present but CTcry is not. If the ratio is between the thresholds R1 and R2, this indicates that both CTcry and NGopa are present. If neither CTcry nor NGopa is present in the reaction (Figure 9A), there is no need to calculate the ratio, and it is indicated as N / A, as shown in Figure 9B. [Figure 10]PCR amplification curves obtained at 52°C in the HEX (A-D) and FAM (E-H) channels for various targets are shown. PCR curves shown as dashed lines represent the presence of a single gene target per reaction, while PCR curves shown as solid lines represent the presence of both gene targets at 20,000 copies (black line) and 32 copies (gray line) of the target per reaction. In the HEX channel, results are shown for CTcry only (A), CTcry and NGopa (B), NGopa only (C), and all remaining off-target controls (D), including 10,000 copies of TFRC (genomic DNA endogenous control), TVbtub, and MgPa. In the FAM channel, results are shown for TVbtub only (E), TVbtub and MgPa (F), MgPa only (G), and all remaining off-target controls (H), including 10,000 copies of TFRC (genomic DNA endogenous control) or 20,000 copies and 32 copies of CTcry and NGopa. In all figures, the dashed black line represents the no-template control (NF HO). Amplification curves are the average of fluorescence levels from triplicate reactions. [Figure 11] Figure 1 shows the signal change (ΔS, where ΔS = SD-post-PCR-SD-pre-PCR) for detecting (A) CTcry and NGopa in HEX and (B) TVbtub and MgPa in FAM using endpoint assay method 1. Results are presented in black and white for ΔSD1 (52°C) and gray for ΔSD2 (70°C). Values ​​for each sample are the average of three replicates, and error bars represent the standard deviation between these replicates. [Figure 12] Qualitative endpoint detection of NGopa in the HEX channel (A) and MgPa in the FAM channel (B) is shown (ΔSD2-ΔSD1=ΔΔSD2ΔSD1) using endpoint analysis method 2. Values ​​for each sample are the average of three replicates, and error bars represent the standard deviation among these replicates. [Figure 13]The changes in fluorescence signal obtained in the HEX (Figures 13A and 13B) and FAM (Figures 13C and 13D) channels at two different temperatures using endpoint assay 3 are shown. (Figure 13A) Detection of CTcry (CT, 20,000 (20K) or 32 copies) and / or NGopa (NG, 20,000 (20K) or 32 copies) using ΔSD2 in the HEX channel. (Figure 13B) Differentiation of CTcry and NGopa in HEX using the ratio ΔSD1:ΔSD2. If ΔSD1:ΔSD2 > threshold R1, detection of CTcry alone is determined. If ΔSD1:ΔSD2 < threshold R2, detection of NGopa alone is determined. If ΔSD1:ΔSD2 > threshold R2 and < threshold R1, detection of coinfection containing both targets is determined. (Figure 13C) Detection of TVbtub and / or MgPa using ΔSD2 in the FAM channel, (Figure 13D) Differentiation of TVbtub and MgPa in FAM using the ratio ΔSD1:ΔSD2. If ΔSD1:ΔSD2 > threshold R1, detection of TVbtub alone is determined. If ΔSD1:ΔSD2 < threshold R2, detection of MgPa alone is determined. If ΔSD1:ΔSD2 > threshold R2 and < threshold R1, detection of coinfection containing both targets is determined. Values ​​for each sample are the average of three replicates, and error bars represent the standard deviation between these replicates. [Figure 14]Figure 14A shows the detection of TFRC (Figure 14A) at temperature 1 (D1) and TPApolA (Figure 14B) at temperature 2 (D2) in the Cy5.5 channel using endpoint assay method 2. Detection of TFRC (Figure 14A) is achieved by subtracting pre-PCR fluorescence at temperature 1 from post-PCR fluorescence at temperature 1 (SD1). Detection of TPApolA (Figure 14B) is achieved by subtracting pre-PCR fluorescence at temperature 2 from post-PCR fluorescence at temperature 2 (ΔSD2) followed by subtracting ΔSD1 (ΔΔSD2ΔSD1). Values ​​for each sample are the average of two replicates: NF HO, 10,000 cps TPApolA, 40 cps TPApolA, 10,000 cps TPApolA and 10,000 cps TFRC, and 40 cps TPApolA and 10,000 cps TFRC. The value of 10,000 cps TFRC is the average of 48 replicates, as TFRC was used as an endogenous control and was present in genomic DNA at 10,000 cps in each reaction well, except for the TPApolA-only sample. Error bars represent the standard deviation between each replicate for each sample. [Figure 15] (Figure 15A) Melting profiles generated by cleavage of Substrate 4 in the presence of 10,000 copies of TFRC (solid black line). (Figure 15B) Melting profiles generated by cleavage of LOCS-3 in the presence of 10,000 copies of TPApolA (solid black line) and 40 copies of TPApolA (solid gray line). (C) Melting profiles generated by cleavage of Substrate 4 and LOCS-3 in the presence of 10,000 copies of TPApolA plus 10,000 copies of TFRC (solid black line) and 40 copies of TPApolA plus 10,000 copies of TFRC (solid gray line). Melting profiles resulting from the absence of both targets (NF HO) are represented using dashed lines (Figures 15A-C). Results represent the rate of change in fluorescence with temperature (-d(RFU) / dT). [Figure 16]PCR amplification plots obtained from reactions containing 20,000 copies of CTcry (solid black line), 20,000 copies of NGopa (dashed black line), 20,000 copies of both targets (solid gray line), or neither target (NTC, dashed gray line) at 39°C (A) and 72°C (B) are shown. Thresholds X and Y are indicated for amplification plots obtained at 39°C and 72°C, respectively. End-point fluorescence values ​​designated EX1, EX2, EY1, and EY2 are shown. [Figure 17] PCR amplification plots obtained from reactions containing 0 copies (solid black line), 32 copies (gray line), or 20,000 copies (dotted black line) of target X / CTcry in a background of target Y / NGopa at various copy numbers as indicated. Plots A, D, and G show the fluorescence at 39°C for reactions containing 20,000 copies (A), 32 copies (D), or no copies (G) of NGopa. Plots B, E, and H show the fluorescence at 74°C for reactions containing 20,000 copies (B), 32 copies (E), or no copies (H) of NGopa. Plots C, F, and I show the fluorescence at 74°C, after normalization to FAF, for reactions containing 20,000 copies (C), 32 copies (F), or no copies (I) of NGopa. [Figure 18] PCR amplification curves for quantitative detection of human GAPDH at 52°C (D1) in the FAM channel. (Figure 18A) The curves represent signals generated from 10,000 copies (gray solid line) and 100 copies (black dashed line) of the GAPDH target alone. (Figure 18B) The results are from 10,000 copies (gray solid line) and 100 copies (black dashed line) of the MgPa target alone. (Figure 18C) The curves represent signals generated from target mixtures containing 10,000 copies (gray solid line) and 100 copies (black dashed line) of GAPDH and MgPa, respectively. The no-template control (NF HO) is represented as a solid black line in (Figures 18A-C). Amplification curves are the average of fluorescence levels from triplicate reactions. [Figure 19]Figure 19 shows the qualitative detection of GAPDH and MgPa using one TaqMan probe and one LOCS probe, respectively, at two temperatures in the FAM channel. Results were obtained using endpoint assay methods 1 to 3. Values ​​for each sample are the average of three replicates, and error bars represent the standard deviation between these replicates. (Figure 19A) Results obtained with endpoint assay method 1 show the signal change (ΔS) between post-PCR and pre-PCR fluorescence measurements. Results are represented in black and white for ΔSD1 (52 °C) and gray for ΔSD2 (70 °C). (Figure 19B) Detection of MgPa alone using results obtained from endpoint assay method 2 (ΔΔSD2ΔSD1). (Figures 19C and 19D) Results obtained from endpoint assay method 3 (ΔSD1:ΔSD2). (Figure 19C) Detection of GAPDH (human 10,000 or 100 copies) and / or MgPa (MG 10,000 or 100 copies) using ΔSD2 in the FAM channel. (Figure 19D) Differentiation of GAPDH and MgPa in FAM using the ratio of ΔSD1:ΔSD2. If ΔSD1:ΔSD2 > threshold R1, detection of GAPDH alone is determined. If ΔSD1:ΔSD2 < threshold R2, detection of MgPa alone is determined. If ΔSD1:ΔSD2 > threshold R2 and < threshold R1, detection of coinfection containing both targets is determined. Results are measured in ratio units. [Figure 20] PCR amplification curves obtained in the FAM channel from reactions containing either 25,600 copies of TVbtub (dotted black line), 25,600 copies of MgPa (dashed black line), a mixture containing 25,600 copies of both targets (solid gray line), or no target (NF HO, solid black line) at 52°C (Figure 20A) and 74°C (Figure 20B). The increase in fluorescence at 52°C (D1) indicates the presence of TVbtub as detected by the molecular beacon, and the increase in fluorescence at 74°C (D2) indicates the presence of MgPa as detected by the LOCS probe. The Cq values ​​determined at D1 and D2 were used to quantify the amount of TVbtub and MgPa in the samples, respectively, without the need for special analytical methods. The curves represent the average fluorescence levels from triplicate reactions. [Figure 21]Shown are the standard curves obtained at 52°C (D1) used to quantify TVbtub (Figure 20A) and 74°C (D2) used to quantify MgPa (Figure 20B). Triplicates of 25600, 6400, 1600, 400, and 100 copies of synthetic TVbtub and MgPa G-Block templates were used to generate the standard curves. [Figure 22]

[0033] Figure 1 illustrates an embodiment in which a dual hybridization probe can be combined with a LOCS probe that can be cleaved by an MNAzyme. Both the dual hybridization probe and the LOCS probe can be labeled with the same fluorophore. The two dual hybridization probes can be capable of binding to target 1 at Tm A and Tm B, respectively. These can be combined with an intact LOCS probe that can have a stem region with Tm C and a loop region that can be cleaved by an MNAzyme in the presence of a second target 2, thus generating a split LOCS probe with Tm D, which is less than Tm C. The presence of target 1 and / or target 2 can be determined by measuring fluorescence at two temperatures in real time, or by using separate measurements taken at or near the start of amplification and after amplification. [Figure 23] Figure 23A shows simultaneous endpoint detection of two targets (TVbtub and MgPa) in the FAM channel using one non-cleavable molecular beacon and one LOCS probe by measuring the change in fluorescence during PCR at 52°C (ΔSD1, Figure 23A) and 74°C (ΔSD2, Figure 23B). Plotted values ​​are the average of triplicate reactions containing various amounts of TVbtub and / or MgPa, as designated on the graph. In Figure 23A, ΔSD1 above the designated threshold indicates the presence of TVbtub in the reaction, or below indicates its absence. In Figure 23B, ΔSD2 above the designated threshold indicates the presence of MgPa in the reaction, or below indicates its absence. The Y-axis is the determined increase in fluorescence at 52°C (ΔSD1, Figure 23A) or 74°C (ΔSD2, Figure 23B). [Figure 24]Figure 24A shows simultaneous endpoint detection of two targets (TVbtub and MgPa) in the FAM channel using one non-cleavable molecular beacon and one LOCS probe by measuring the change in calibrated fluorescence signal during PCR at 52°C (ΔSD1 / C, Figure 24A) and 74°C (ΔSD2 / C, Figure 24B). Plotted values ​​are the average of replicates containing various amounts of TVbtub and / or MgPa template, as designated on the graph, and error bars represent the standard deviation among these replicates. In Figure 24A, ΔSD1 / C above threshold 1 indicates the presence of TVbtub in the reaction, or below indicates its absence. In Figure 24B, ΔSD2 / C above threshold 2 indicates the presence of MgPa in the reaction, or below indicates its absence. [Figure 25] Figure 25 shows simultaneous endpoint detection of two targets (CTcry and NGopa) in the HEX channel using one linear MNAzyme substrate and one LOCS probe by measuring the change in fluorescent signal during PCR at 52°C (ΔSD1, Figure 25A) and 70°C (ΔSD2, Figure 25B) and the calibrated signal at 52°C (ΔSD1 / C, Figure 25C) and 70°C (ΔSD2 / C, Figure 25D) across three Bio-Rad CFX96 instruments tested (instrument 1 with black stripes, instrument 2 with gray stripes, and instrument 3 with white stripes). Plotted values ​​are the average of triplicates containing varying amounts of CTcry and / or NGopa template, as designated on the graph, and error bars represent the standard deviation among these replicates. A signal in Figure 25A or a calibrated signal in Figure 25C above threshold C1 indicates the presence of CTcry in the reaction, or below indicates its absence. The calibrated signal in Figure 25D indicates the absence of CTcry and NGopa below threshold C2, the presence of both CTcry and NGopa above threshold C3, and the presence of only one of CTcry and NGopa between thresholds C2 and C3. Figure 25B shows that the value of threshold C3 differs from that shown in Figure 25D and varies between instruments without calibration (threshold C2 is not shown in Figure 25B). [Figure 26]Figure 26 shows simultaneous endpoint detection of target 1 (CTcry) and / or target 2 (NGopa) in the HEX channel using one linear MNAzyme substrate and one LOCS probe by using endpoint analysis method 2. By measuring post-PCR signals obtained from experimental samples at D1 (52°C) and D2 (70°C), and determining background signals as pre-PCR fluorescence measurements (SD3) from the same reaction wells at 40°C (Figures 26A-B), 52°C (Figures 26C-D), and 62°C (Figures 26E-F), graphs showing NSD1 (LHS) and ΔNSD2NSD1 (RHS) were determined for CTcry and NGopa detection, respectively. In Figures 26A, 26C, and 26E, when NSD1 is above threshold X1, this indicates the presence of CTcry in the reaction, and when it is below, it indicates its absence. In Figures 26B, 26D and 26F, if ΔNSD2NSD1 is above the threshold X2, this indicates the presence of NGopa in the reaction, or below it indicates its absence. [Figure 27] Figure 27 shows simultaneous endpoint detection of two targets (CTcry and NGopa) in the HEX channel using one linear MNAzyme substrate and one LOCS probe by using endpoint analysis method 2. NSD1(LHS) and ΔNSD2NSD1(RHS) were determined for CTcry and NGopa detection, respectively, by measuring the post-PCR signal acquired at D1 (52°C) and D2 (70°C) and determining the background signal as the pre-PCR fluorescence measurement (SD3) as the average of the no-template control signals measured at D1 / D3B before PCR (Figures 27A-B) and at D1 and D2 before PCR (Figures 27C-D) and after PCR (Figures 27E-F). In Figures 27A, 27C, and 27E, when NSD1 is above threshold X1, it indicates the presence of CTcry in the reaction, and when it is below, it indicates its absence. In Figures 26B, 26D and 26F, if ΔNSD2NSD1 is above the threshold X2, this indicates the presence of NGopa in the reaction, or below it indicates its absence.

[0232] definition As used in this application, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the phrase "a polynucleotide" also includes a plurality of polynucleotides.

[0233] As used herein, the term "comprising" means "including." Variations of the word "comprising," such as "comprise" and "comprises," have correspondingly different meanings. Thus, for example, a polynucleotide that "comprises" a sequence of nucleotides may consist solely of that sequence of nucleotides or may include one or more additional nucleotides.

[0234] As used herein, the term "plurality" means two or more. In certain specific aspects or embodiments, plurality can 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.

[0235] As used herein, the term "subject" includes any animal of economic, social, or research importance, including bovine, equine, ovine, primate, avian, and rodent species. Thus, a "subject" can be a mammal, such as, for example, a human or a non-human mammal. Microbial subjects are also encompassed, including, but not limited to, bacteria, viruses, fungi / yeast, protozoa, and nematodes. A "subject" according to the present invention also includes infectious agents such as prions.

[0236] As used herein, the terms "polynucleotide" and "nucleic acid" can be used interchangeably and refer to a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases, or analogs, 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 RNA, ribosomal RNA, derivatives thereof, amplicons thereof, or any combination thereof. As a non-limiting example, the source of the nucleic acid can be selected from the group including synthetic, mammalian, human, animal, plant, fungal, bacterial, viral, archaeal, or any combination thereof. It is possible.

[0237] As used herein, the term "oligonucleotide" refers to a segment of DNA or a DNA-containing nucleic acid molecule, or an RNA or an RNA-containing molecule, or a combination thereof. Examples of oligonucleotides include nucleic acid targets, substrates, e.g., those that can be modified by MNAzymes, primers such as those used in in vitro target amplification by methods such as PCR, components of MNAzymes, and various other types of reporter probes, including, but not limited to, TaqMan or hydrolysis probes, molecular beacons, sloppy beacons, eclipse probes, Scorpion uniprobes, Scorpion biprobe primer / probes, capture / pitchers, and dual hybridization probes. The term "oligonucleotide" includes reference to any specific sequence and its complementary sequence, unless otherwise indicated.Oligonucleotides include 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, 2'-O-methylcytidine, 5-carboxymethylaminomethylthiouridine, 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-methyladenosine, 2-methylguanosine, 3-methylcytidine, 5-methylcytidine, N6-methyladenosine, 7-methylguanosine, 5-methylaminomethyluridine, 5-methoxyaminomethyl-2-thiouridine, beta-D-mannosylmethyluridine, 5-methoxycarbonylmethyluridine, 5-methoxyuridine, 2-methylthio-N6-isopenteny Luadenosine, N-((9-beta-ribofuranosyl-2-methylthiopurin-6-yl)carbamoyl)threonine, N-((9-beta-ribofuranosylpurin-6-yl)N-methyl-carbamoyl)threonine, uridine-5-oxyacetic acid methyl ester, uridine-5-oxyacetic acid (v), wibutoxosin, pseudouridine, queosin, 2-thiocytidine, 5-methyl-2-thiouridine, 2-thiouridine, 4 and may contain at least one addition or substitution, including but not limited to the group including -thiouridine, 5-methyluridine, N-((9-beta-D-ribofuranosylpurin-6-yl)carbamoyl)threonine, 2'-O-methyl-5-methyluridine, 2'-O-methyluridine, wybutosine, 3-(3-amino-3-carboxypropyl)uridine, beta-D-arabinosyluridine, beta-D-arabinosylthymidine.

[0238] The terms "polynucleotide" and "nucleic acid" and "oligonucleotide" include reference to any specific sequence and its complementary sequence, unless otherwise indicated.

[0239] As used herein, the terms "complementary," "complementarity," "matching," and "matched" refer to the ability of nucleotides (e.g., deoxyribonucleotides, ribonucleotides, or combinations thereof) to hybridize to each other through non-canonical base pairing, including Watson-Crick base pairing, wobble base pairing, and Hoogsteen base pairing (LNA, PNA, or BNA), or unnatural base pairing (UBP). Bonds can be formed through Watson-Crick base pairing between adenine (A) and uracil (U) bases, between adenine (A) and thymine (T) bases, and between cytosine (C) and guanine (G) bases. A wobble base pair is a non-canonical base pairing between two nucleotides in a polynucleotide duplex (e.g., guanine-uracil, inosine-uracil, inosine-adenine, and inosine-cytosine). Hoogsteen base pairs, like Watson-Crick base pairs, occur between adenine (A) and thymine (T) bases and between cytosine (C) and guanine (G) bases, but the conformation of the purine relative to the pyrimidine is different compared to Watson-Crick base pairing. Unnatural base pairs are manufactured subunits synthesized in the laboratory and do not occur in nature. Nucleotides that are referred to as "complementary" or "complements" of each other are nucleotides that have the ability to hybridize together either by Watson-Crick base pairing between their respective bases or by non-standard base pairing (wobble base pairing, Hoogsteen base pairing) or unnatural base pairing (UBP). As used herein, a sequence of nucleotides that is "complementary" to another sequence of nucleotides can mean that the first sequence is 100% identical 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 to the complement of the second sequence.As used herein, reference to a sequence of nucleotides that is "substantially complementary" to another sequence of nucleotides can mean that the first sequence is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the complement of the 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.

[0240] As used herein, the terms "non-complementary," "not complementary," "mismatched," and "mismatched" refer to nucleotides (e.g., deoxyribonucleotides, ribonucleotides, and combinations thereof) that lack the ability to hybridize together by either Watson-Crick base pairing or wobble base pairing between their respective bases. As used herein, a sequence of nucleotides that is "non-complementary" to another sequence of nucleotides can mean that the first sequence is 0% identical to the complement of the 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.

[0241] As used herein, reference to a sequence of nucleotides that is "substantially non-complementary" to another sequence of nucleotides can mean that the first sequence is at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, or less than 40% identical to the complement of the 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.

[0242] As used herein, the term "target" refers to any molecule or analyte present in a sample that can be detected using the methods of the present invention. The term "target" is understood to include nucleic acid targets as well as non-nucleic acid targets such as, for example, proteins, peptides, analytes, ligands, and ions (e.g., metal ions).

[0243] As used herein, "enzyme" refers to any molecule capable of catalyzing a chemical reaction (e.g., amplifying a polynucleotide, cleaving a polynucleotide, etc.). Non-limiting 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, DNAzymes, and aptazymes. Non-limiting examples of suitable protein enzymes include exonucleases and endonucleases. Enzymes generally provide catalytic activity that aids in carrying out one or more of the methods described herein. As a non-limiting example, exonuclease activity can be, for example, the intrinsic catalytic activity of a polymerase. As a non-limiting example, endonuclease activity can be, for example, the intrinsic catalytic activity of a restriction enzyme, including a nicking endonuclease, a riboendonuclease, or a dual-specific nuclease (DSN).

[0244] As used herein, "amplicon" refers to a nucleic acid (e.g., DNA or RNA, or a combination thereof) that is the product of a natural or artificial nucleic acid amplification or replication event, including, but not limited to, PCR, RT-PCR, SDA, NEAR, HDA, RPA, LAMP, RCA, TMA, LCR, RAM, 3SR, NASBA, and any combination thereof.

[0245] As used herein, the term "stem-loop oligonucleotide" is understood to mean a DNA or DNA-containing molecule, or an RNA or RNA-containing molecule, or a combination thereof (i.e., a DNA-RNA hybrid molecule or complex) that comprises or consists of a double-stranded stem component joined to a single-stranded loop component. The double-stranded stem component comprises a forward strand hybridized to a complementary reverse strand by complementary base pairing, 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 double-stranded stem component may further comprise a detection moiety, including, but not limited to, one or more fluorophores, 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 gold or silver nanoparticles on both strands for colorimetric detection, immobilization of gold nanoparticles on the gold surface of one strand (e.g., the forward strand) and on the opposing strand (e.g., the reverse strand) for SPR detection, and immobilization of methylene blue molecules on the electrode surface of one strand (e.g., the forward strand) and on the opposing strand (e.g., the reverse strand) for electrochemical detection.

[0246] As used herein, the term "stem-loop oligonucleotide" is understood to include a "LOCS," which is also referred to herein as a "LOCS oligonucleotide," "LOCS structure," "LOCS reporter," "intact LOCS," "closed LOCS," and "LOCS probe." The single-stranded loop component of a LOCS may include a region that can function as a substrate for a catalytic nucleic acid, such as, for example, an MNAzyme, DNAzyme, ribozyme, apta-MNAzyme, or aptazyme. Additionally or alternatively, the single-stranded loop component may include a region that is complementary to a target nucleic acid (e.g., a target for detection, quantification, etc.) and / or an amplicon derived therefrom, and further capable of functioning as a substrate for an exonuclease enzyme. As a non-limiting example, exonuclease activity may be an inherent activity of a polymerase enzyme. Additionally or alternatively, the single-stranded loop component region may include (i) a region that may be complementary to a target to be detected, (ii) a region that may contain one strand of a double-stranded restriction enzyme recognition site, and (iii) a region that may function as a substrate for a restriction enzyme, e.g., a nicking endonuclease. As used herein, the terms "split stem-loop oligonucleotide," "split LOCS," "split LOCS oligonucleotide," "split LOCS structure," "split LOCS reporter," "split LOCS probe," "cleaved LOCS," and "degraded LOCS" are used interchangeably herein and are understood to refer to a "LOCS" in which the single-stranded loop component has been cleaved, digested, and / or degraded (e.g., by an enzyme described herein) such that at least one bond between adjacent nucleotides within the loop has been removed, thereby providing a discontinuous section within the loop region. In a split LOCS, the forward and reverse strands of the double-stranded stem portion may retain the ability to hybridize to each other in a temperature-dependent manner to form a stem.

[0247] LOCSs are designed to contain a cleavable loop region, allowing target-dependent cleavage of the loop region by an enzyme to generate a split LOCS. This, in turn, can facilitate detection of the target from a detectable signal generated at a specific temperature after association (hybridization) or dissociation of the stem portion of the intact or split LOCS. In contrast, as used herein, a molecular beacon contains a loop region that is not cleavable during the methods described herein. "LOCS" refers to a stem-loop oligonucleotide designed to contain a target. Molecular beacons can mediate target detection by generating a detectable signal at a specific temperature after the loop portion of the probe associates (hybridizes) or dissociates (separates) with the target to be detected. Thus, the main difference between these two types of stem-loop structures in the context of the present invention is that LOCSs are monitored by measuring the change in signal resulting from hybridization or dissociation of the stem region of an intact or split LOCS, whereas molecular beacons are monitored by measuring the change in signal resulting from hybridization or dissociation of the loop region and the target.

[0248] As used herein, the term "universal stem" refers to a double-stranded sequence that can be incorporated into any LOCS structure. The same "universal stem" can be used in LOCSs containing loops that contain either catalytic nucleic acid substrates or sequences complementary to a target of interest. A single universal stem can be used as a surrogate marker for any target that can facilitate the division of a particular LOCS. A series of universal stems can be incorporated into a series of LOCSs designed to analyze any set of targets.

[0249] As used herein, the term "universal LOCS" refers to a LOCS structure containing a "universal stem" and a "universal loop" that contains a universal catalytic nucleic acid substrate that can be cleaved by any MNAzyme with a complementary substrate-binding arm, regardless of the sequence of the MNAzyme target-sensing arm. A single universal LOCS can be used as a surrogate marker for any target that can facilitate cleavage of a specific LOCS. A series of universal LOCSs can be incorporated into any multiplexed assay designed to analyze any set of targets.

[0250] As used herein, the terms "nucleic acid enzyme," "catalytic nucleic acid," "nucleic acid having catalytic activity," and "catalytic nucleic acid enzyme" are used interchangeably herein and refer to a DNA or DNA-containing molecule or complex, or an RNA or RNA-containing molecule or complex, or a combination thereof (i.e., a DNA-RNA hybrid molecule or complex) that can recognize at least one substrate and catalyze the modification (e.g., cleavage) of the at least one substrate. The nucleotide residues of a catalytic nucleic acid can include the bases A, C, G, T, and U, as well as derivatives and analogs thereof. The above terms include unimolecular nucleic acid enzymes that can include 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, that is a DNA-RNA hybrid molecule that can recognize at least one substrate and catalyze the modification (e.g., cleavage) of the at least one substrate. The above terms include nucleic acid enzymes comprising DNA or DNA-containing complexes, or RNA or RNA-containing complexes, or combinations thereof, that are DNA-RNA hybrid complexes capable of recognizing at least one substrate and catalyzing the modification (such as cleavage) of that at least one substrate. The terms "nucleic acid enzyme", "catalytic nucleic acid", "catalytically active nucleic acid", and "catalytic nucleic acid enzyme" include within their meaning MNAzymes.

[0251] As used herein, the terms "MNAzyme" and "multi-component nucleic acid enzyme" have the same meaning and refer to two or more oligonucleotide sequences (e.g., partzymes) that form an active nucleic acid enzyme that can catalytically modify a substrate only in the presence of an MNAzyme assembly facilitator (e.g., a target). An "MNAzyme" is also known in the art as a "PlexZyme." MNAzymes can catalyze a series of reactions including cleavage of the substrate, as well as other enzymatic modifications of the substrate(s). MNAzymes with endonuclease or cleavage activity are also known as "MNAzyme cleaving agents." It is known that the constituent partzymes, partzymes A and B, each bind to an assembly facilitator (e.g., a target DNA or RNA sequence) through base pairing. An MNAzyme is formed only when the sensor arms of partzymes A and B hybridize adjacent to each other on the assembly facilitator. The substrate arm of the MNAzyme engages the substrate, and its modification (e.g., cleavage) is catalyzed by the catalytic core of the MNAzyme, formed by the interaction of the partial catalytic domains of partzymes A and B. MNAzymes can cleave DNA / RNA chimeric reporter substrates. MNAzyme cleavage of the substrate between a fluorophore and quencher dye pair can generate a fluorescent signal. The terms "multicomponent nucleic acid enzyme" and "MNAzyme" include bipartite structures composed of two molecules, or tripartite structures composed of three nucleic acid molecules, or other multipartite structures, such as those formed by four or more nucleic acid molecules.

[0252] As used herein, the terms "MNAzyme" and "multi-component nucleic acid enzyme" are understood to encompass all known MNAzymes and modified MNAzymes, including those disclosed in any one or more of PCT Patent Publication Nos. WO / 2007 / 041774, WO / 2008 / 040095, WO2008 / 122084, and related US Patent Publication Nos. 2007-0231810, 2010-0136536, and 2011-0143338 (the contents of each of these documents are incorporated herein by reference in their entirety). Non-limiting 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, and multi-component nucleic acid inactive proenzymes (MNAi), each of which are described in detail in one or more of WO / 2007 / 041774, WO / 2008 / 040095, US2007-0231810, US2010-0136536, and / or US2011-0143338.

[0253] As used herein, the terms "partzyme," "constituent partzyme," and "partzyme component" refer to DNA-containing, RNA-containing, or DNA-RNA-containing oligonucleotides, two or more of which can come together to form an "MNAzyme" only in the presence of an MNAzyme assembly facilitator, as defined herein. In certain preferred embodiments, one or more, and preferably at least two, constituent partzymes may comprise three regions or domains: a "catalytic" domain that forms part of the catalytic core that catalyzes the modification; a "sensor arm" domain that can associate with and / or bind to an assembly facilitator; and a "substrate arm" domain that can 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 a partzyme that binds to an assembly facilitator, e.g., a target. A partzyme 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 shortened sensor arm, and a stabilising arm component that stabilizes the MNAzyme structure by interacting with either the assembly facilitator or the substrate.

[0254] As used herein, the terms "assembly facilitator molecule," "assembly facilitator," "MNAzyme assembly facilitator molecule," and "MNAzyme assembly facilitator" refer to an entity that can promote the self-assembly of component partzymes to form a catalytically active MNAzyme by interacting with the sensor arm of an MNAzyme. As used herein, an assembly facilitator may promote the assembly of MNAzymes that have cleavage or other enzymatic activity. In a preferred embodiment, an assembly facilitator is required for the self-assembly of an MNAzyme. An assembly facilitator may be comprised of a single molecule, or may be comprised of two or more "assembly facilitator components" that pair with or bind to the sensor arms of one or more oligonucleotide "partzymes." An assembly facilitator may comprise one or more nucleotide components that do not share sequence complementarity with the sensor arms of an MNAzyme. An 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 RNA, ribosomal RNA, their derivatives, amplicons, or any combination thereof. The nucleic acid may be amplified. The amplification may include one or more of PCR, RT-PCR, SDA, NEAR, HDA, RPA, LAMP, RCA, TMA, RAM, LCR, 3SR, or NASBA.

[0255] MNAzymes can cleave linear substrates and / or substrates present within the loop region of a stem-loop LOCS reporter probe structure. Cleavage of the linear substrate can separate the fluorophore and quencher, allowing for target detection. Cleavage of the loop region of the LOCS by an MNAzyme can generate a split-LOCS structure composed of two fragments that remain hybridized and can associate at temperatures below the melting temperature of the stem, and can separate and dissociate at temperatures above the melting temperature of the stem of the split-LOCS.

[0256] The terms "detectable effect" and "detectable signal" are used interchangeably herein and are understood to have the same meaning. This term refers to a signal or effect produced from a detection moiety that is attached to or otherwise associated with an oligonucleotide of the invention (e.g., a probe, reporter, or substrate), typically after modifying the oligonucleotide to change its conformation, structure, orientation, position relative to other entities, etc. The modification can be induced, for example, by the presence of a target that the oligonucleotide is designed to detect. Non-limiting examples of such modifications (e.g., induced by the presence of a target) include the opening of the stem-loop portion of a molecular beacon, the opening of the double-stranded portion of a Scorpion uniprobe and biprobe, binding of a dual hybridization probe to a target sequence, the generation of a catcher duplex structure, and cleavage / digestion of a linear MNAzyme substrate or TaqMan probe, etc. The detectable effect can be detected by a variety of methods, including fluorescence spectroscopy, surface plasmon resonance (SPR), mass spectroscopy, NMR, electron spin resonance, polarized fluorescence spectroscopy, circular dichroism spectroscopy, immunoassays, chromatography, radiometric measurements, photometry, scintigraphy, electronic methods, electrochemical methods, UV, visible, or infrared spectroscopy, enzymatic methods, or any combination thereof. The detectable signal / effect can be detected or quantified, and its magnitude can indicate the presence and / or amount of an input, such as the amount of a target molecule present in a sample. Furthermore, the magnitude of the detectable signal / effect provided by the detection moiety can be adjusted by varying the conditions of the reaction in which the oligonucleotide containing the detectable moiety is utilized, including, but not limited to, the reaction temperature. Thus, the ability of the detectable moiety to bind to or otherwise associate with the oligonucleotide to generate a target-dependent signal and / or a target-independent background signal can be adjusted.

[0257] As used herein, the terms "background signal" and "baseline signal" are used interchangeably and are understood to have the same meaning. This term refers to a signal generated by a detectable moiety bound to or otherwise associated with an oligonucleotide of the invention, regardless of the presence or absence of the particular target that the oligonucleotide is designed to measure or detect under particular measurement conditions.

[0258] As used herein, the terms "polynucleotide substrate," "oligonucleotide substrate," and "substrate" include any single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases, or analogs, derivatives, variants, fragments, or combinations thereof, that can be recognized, acted upon, or modified by an enzyme, including a catalytic nucleic acid enzyme. A "polynucleotide substrate" or "oligonucleotide substrate" or "substrate" can be modified by various enzymatic activities, including, but not limited to, cleavage. Cleavage or degradation of a "polynucleotide substrate" or "oligonucleotide substrate" or "substrate" can provide a "detectable effect" for monitoring the catalytic activity of an enzyme. A "polynucleotide substrate" or "substrate" can be capable of cleavage or degradation by one or more enzymes, including, but not limited to, catalytic nucleic acid enzymes such as MNAzymes, apta-MNAzymes, DNAzymes, aptazymes, ribozymes, and / or protein enzymes such as exonucleases or endonucleases.

[0259] As used herein, a "reporter substrate" is a substrate specifically adapted to facilitate measurement of either the cleavage or degradation of the substrate or the appearance of the cleaved product in the context of a catalytic reaction. The reporter substrate may be free in solution or bound (or "tethered") to, for example, a surface or another molecule. The reporter substrate may be labeled by any of a variety of means, including, for example, a fluorophore (with or without one or more additional components such as a quencher), a radiolabel, biotin (e.g., biotinylation), or a chemiluminescent label.

[0260] As used herein, a "linear MNAzyme substrate" is a substrate, e.g., a reporter substrate, that is recognized and catalytically acted on by multiple MNAzymes. A "linear MNAzyme substrate" does not contain a sequence at its 5' or 3' end that can hybridize to form a stem. Alternatively, the MNAzyme substrate may be present within the loop region of a LOCS probe.

[0261] As used herein, a "universal substrate" is a substrate, e.g., a reporter substrate, that is recognized and catalytically acted upon by multiple MNAzymes, each capable of recognizing a different assembly facilitator. The use of such a substrate facilitates the development of distinct assays for detecting, identifying, or quantifying various assembly facilitators using structurally related MNAzymes, all of which recognize the universal substrate. Each of these universal substrates can be independently labeled with one or more labels. In preferred embodiments, one or more universal substrates are labeled with independently detectable labels, allowing for the creation of a convenient system for independently or simultaneously detecting various assembly facilitators using MNAzymes. In some embodiments, the substrate is capable of catalytic modification by a DNAzyme that is catalytically active in the presence of a cofactor, e.g., a metal ion cofactor such as lead or mercury. In some embodiments, the substrate may be suitable for catalytic modification by an aptazyme that can 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 capability of the nucleic acid enzyme portion.

[0262] As used herein, the terms "probe" and "reporter" refer to oligonucleotides used to detect target molecules (e.g., nucleic acids or analytes). Non-limiting examples of standard probes or reporters known in the art include, but are not limited to, linear MNAzyme substrates, TaqMan or hydrolysis probes, molecular beacons, sloppy beacons, eclipse probes, Scorpion uniprobes, Scorpion biprobe primer / probes, capture / pitcher oligonucleotides, and dual hybridization probes. Embodiments of the present invention combine standard probes and LOCS probes. Some LOCS probes contain a nucleic acid enzyme substrate within a loop region that may be universal, which is capable of catalytic cleavage by nucleic acid enzymes such as MNAzymes, DNAzymes, and aptazymes. Other LOCS probes contain target-specific sequences within the loop region that are capable of catalytic cleavage by protein enzymes, including endonucleases and exonucleases.

[0263] The term "product" refers to the new molecule(s) generated as a result of enzymatic modification of a substrate. As used herein, the term "cleavage product" refers to the new molecule(s) generated as a result of enzymatic cleavage or endonuclease activity. In some embodiments, the products of enzymatic cleavage or degradation of an intact LOCS structure comprise two oligonucleotide fragments, collectively referred to as a split-LOCS, which may be capable of either hybridization or dissociation / separation depending on the temperature of the reaction.

[0264] As used herein, the use of the terms "melting temperature" and "Tm" in the context of polynucleotides, unless specifically indicated otherwise, is understood to refer to the melting temperature (Tm) calculated using Wallace's rule, whereby Tm = 2°C (A + T) + 4°C (G + C) (see Wallace et al., (1979) Nucleic Acids Res. 6, 3543). The effect of sequence composition on melting temperature can be understood using the nearest neighbor method governed by the following formula: Tm(°C) = ΔH° / (ΔS° + R ln[oligo]) - 273.15. In addition to stem length and sequence composition, other factors known to affect melting temperature include ionic strength and oligonucleotide concentration. Higher oligonucleotide and / or ionic concentrations increase the likelihood of duplex structure formation, leading to increased melting temperatures. In contrast, lower oligonucleotide and / or ionic concentrations favor stem dissociation, leading to decreased melting temperatures.

[0265] As used herein, the term "quencher" includes any molecule that, when in proximity to a fluorophore, absorbs the emission energy generated by the fluorophore and either dissipates this energy as heat or emits light at a wavelength longer 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), and Black Berry quenchers (BBQ).

[0266] As used herein, the term "base" when used in the context of nucleic acids is understood to have the same meaning as the term "nucleotide."

[0267] As used herein, the term "blocker" or "blocker molecule" refers to any molecule or functional group that can be incorporated into an oligonucleotide to prevent a polymerase from using a portion of the oligonucleotide as a template for synthesis of a complementary strand. As a non-limiting example, a hexaethylene glycol blocker can be incorporated into, for example, a Scorpion probe to link its 5' probe sequence to its 3' priming sequence, where the blocker functions to prevent a polymerase from using the probe sequence as a template.

[0268] As used herein, the terms "normalize," "normalizing," and "normalized" refer to the conversion of a measured signal (e.g., a detectable signal produced by a detection moiety) to a scale relative to a known and repeatable value or to a control value.

[0269] As used herein, the term "kit" refers to any delivery device for delivering materials. The term "kit" refers to a delivery system. Such delivery systems include systems that allow for the storage, transport, or delivery of reaction reagents (e.g., labels in appropriate containers, reference samples, supporting materials, etc.) and / or supporting materials (e.g., buffers, instructions for performing an assay, etc.) from one location to another. For example, a kit may include one or more enclosures, such as a box, containing the relevant reaction reagents and / or supporting materials. The term "kit" includes both fragmented and combined kits.

[0270] As used herein, the term "fragmented kit" refers to a delivery system that includes two or more separate containers, each containing a subportion of a component of the overall kit. The containers can be delivered to the intended recipient together or separately. Any delivery system that includes two or more separate containers, each containing a subportion of a component of the overall kit, is included within the meaning of the term "fragmented kit."

[0271] 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., a single box housing each of the desired components).

[0272] As used herein, the use of the term "about" in reference to a recited numerical value is understood to include the recited numerical value and numerical values ​​within ±10 percent of the recited value.

[0273] As used herein, the use of the term "between" when referring to a range of numerical values ​​is understood to encompass each endpoint of the range. For example, a polypeptide between 10 and 20 residues in length includes a polypeptide that is 10 residues long and a polypeptide that is 20 residues long.

[0274] Any statement in this specification about prior art documents, or descriptions in this specification that are derived from or based on those documents, is not an admission that those documents or descriptions from which they are derived are part of the common general knowledge in the relevant art.

[0275] Unless otherwise stated, for purposes of description, all documents referred to herein are hereby incorporated by reference in their entirety.

[0276] Abbreviation The following abbreviations are used herein and throughout the specification: LOCS: Loops attached to stems MNAzyme: multicomponent nucleic acid enzyme, Partzyme: a partial enzyme containing an oligonucleotide; PCR: polymerase chain reaction; gDNA: genomic DNA NTC: No template control qPCR: Real-time quantitative PCR Ct: threshold cycle Cq: quantification cycle R 2 :Correlation coefficient nM: nanomolar mM: millimolar μL: microliter μM: micromolar dNTP: deoxyribonucleotide triphosphate NF-H2O: nuclease-free water; LNA: Locked Nucleic Acid; F: fluorophore, Q: Quencher, N=A, C, T, G, or any analog thereof; N' = any nucleotide that is complementary to or can base pair with N. (N) x : any number N, (N') x : any number N', W:A or T, R: A, G, or AA, rN: any ribonucleotide base, (rN) x : any number rN, rR:A or G, rY:C or U, M: A or C, H: A, C, or T; D: G, A, or T, JOE or 6-JOE: 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein, FAM or 6-FAM: 6-carboxyfluorescein, BHQ1: Black Hole Quencher 1 BHQ2: Black Hole Quencher 2 RT-PCR: reverse transcription polymerase chain reaction SDA: Strand Displacement Amplification NEAR: Nicking enzyme amplification reaction HDA: helicase-dependent amplification RPA: Recombinase Polymerase Amplification LAMP: Loop-mediated isothermal amplification RCA: Rolling Circle Amplification TMA: transcription-mediated amplification 3SR: self-sustaining sequence replication NASBA: Nucleic Acid Sequence-Based Amplification LCR: ligase chain reaction RAM: Branch amplification method IB: Iowa Black® FQ IBR: Iowa Black® RQ shRNA: small hairpin RNA siRNA: small interfering RNA mRNA: messenger RNA tRNA: transfer RNA snoRNA: nucleolar small RNA stRNA: small transient RNA smRNA: small regulatory regulatory RNA Pre-microRNA: precursor microRNA Pri-microRNA: Primary microRNA LHS: Left side RHS: Right side DSO: double-stranded oligonucleotide Tm: melting temperature RFU: relative fluorescence units CT: Chlamydia trachomatis NG: Neisseria gonorrhoeae SPR: surface plasmon resonance GNP: Gold nanoparticles DETAILED DESCRIPTION OF THE INVENTION

[0277] The following detailed description conveys exemplary embodiments of the present invention in sufficient detail to enable those skilled in the art to practice the invention. Features or limitations of the various embodiments described do not necessarily limit other embodiments of the invention or the invention as a whole. Accordingly, the following detailed description does not limit the scope of the present invention, which is defined solely by the claims.

[0278] The present invention relates to methods and compositions for improved multiplexed detection of targets (e.g., nucleic acids, proteins, analytes, compounds, molecules, etc.) The methods and compositions each employ combinations of LOCS oligonucleotides with other oligonucleotide reporters, probes, or substrates that can be used in combination with a variety of other agents.

[0279] -Reporters, probes and substrates According to the present invention, multiplexed detection of target molecules is facilitated using LOCS in combination with other oligonucleotides suitable for use as probes in multiplexed detection assays.

[0280] Many oligonucleotides for the detection of nucleic acid targets have been described and are well known in the art. Suitable oligonucleotides that can be used in combination with LOCS include, but are not limited to, MNAzyme substrates, TaqMan or hydrolysis probes, molecular beacons, sloppy beacons, eclipse probes, Scorpion uniprobes, Scorpion biprobes, dual hybridization probes, and capture / pitcher probes.

[0281] In some embodiments, these oligonucleotides bind directly to targets or target amplicons to facilitate their detection, although MNAzyme substrate and capture / pitcher oligonucleotides provide an exception as they are universal and may be suitable for the detection of any target.

[0282] In some embodiments, the oligonucleotide generates fluorescence in the presence of the target due to enzyme-mediated cleavage or degradation, for example, MNAzyme substrates and TaqMan or hydrolysis probes.

[0283] In other embodiments, the oligonucleotides provide different levels of fluorescent signal as a result of conformational changes induced by binding to a target or target amplicon (e.g., molecular beacons, sloppy beacons, eclipse probes, Scorpion uniprobes, Scorpion biprobes, and dual hybridization probes).

[0284] In the TOCE system, the Catcher changes fluorescence as a result of a conformational change induced by the binding and elongation of the Pitcher that is activated and released only in the presence of the target.

[0285] Any or all of these types of reporter oligonucleotides are suitable for use in conjunction with LOCS probes to mediate detection of multiple targets by measuring changes associated with a single detection moiety, including but not limited to changes in fluorescence measured at a single wavelength.

[0286] Oligonucleotides for combining with LOCS were synthesized according to standard protocols. For example, they can be synthesized by phosphoramidite chemistry using nucleoside and non-nucleoside phosphoramidites in sequential synthesis cycles involving protecting group removal, phosphoramidite coupling, capping, and oxidation, either in solid phase or solution phase, and optionally in an automated synthesizer device. Alternatively, they may be purchased from commercial sources. Non-limiting examples of commercial sources from which MNAzyme substrates, TaqMan or hydrolysis probes, molecular beacons, sloppy beacons, eclipse probes, Scorpion uniprobes, Scorpion biprobes, dual hybridization probes, and capture / pitcher probes can be purchased or otherwise obtained include:MNAzyme 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 and sloppy beacons can be purchased from Integrated DNA Technologies (www.idtdna.com), Eurofins (www.eurofinsgenomics.com), Sigma Aldrich (www.sigmaaldrich.com), and TriLink BioTechnologies (www.trilinkbiotech.com), Eclipse probes can be purchased from Integrated DNA Technologies (www.idtdna.com), and Scorpion uniprobes can be purchased from Sigma. Scorpion bioprobes can be purchased from Bio-Synthesis (https: / / www.biosyn.com), dual hybridization probes can be purchased from Bio-Synthesis (https: / / www.biosyn.com), Sigma Aldrich (www.sigmaaldrich.com) and Eurofins (www.eurofinsgenomics.com), and the Catcher-Pitcher assay can be purchased from Seegene (www.seegene.com).

[0287] -LOCS oligonucleotides An exemplary LOCS oligonucleotide for use in the present invention is shown in Figure 1. The exemplary intact LOCS oligonucleotide shown (Figure 1A, LHS) has a loop region, a stem region, and a fluorophore (F) / quencher (Q) dye pair. While exemplified with a fluorophore / quencher pair, one of skill in the art will recognize that any other suitable detection moiety can be used for the same purpose. The loop region contains a substrate region suitable for enzymatic cleavage or degradation in the presence of a target or target amplicon. Cleavage or degradation of the loop within the intact LOCS generates a split LOCS duplex structure (Figure 1B, RHS).

[0288] In some embodiments, the melting temperature (“Tm”) of the intact LOCS oligonucleotide is higher than the Tm of the split LOCS structure.

[0289] Because intramolecular bonds are stronger than intermolecular bonds, the stem region of an intact LOCS structure generally melts at a higher temperature than the stem of a split, cleaved, or degraded LOCS oligonucleotide structure. For example, the stem of an intact LOCS A melts at a higher Tm A than the Tm B at which the split LOCS stem melts (FIG. 1B). The presence of fluorescence at a temperature that allows melting of the split LOCS but not the intact LOCS indicates the presence of the target or target amplicon. In the exemplary LOCS shown in FIG. 2, the sequence of the loop region of the LOCS oligonucleotide can be, for example, a substrate for an MNAzyme or other catalytic nucleic acid.

[0290] An exemplary LOCS suitable for use in the present invention may contain a loop region comprising a substrate for a catalytic nucleic acid, as shown in FIG. 2. In these embodiments, the LOCS oligonucleotide comprises a universal substrate that can be used to detect any target. The LOCS oligonucleotide contains a stem region, a fluorophore quencher / dye pair (alternative detection moieties described herein can be used), and an intervening loop region comprising a universal substrate for a catalytic nucleic acid, such as an MNAzyme. MNAzymes may detect targets directly or may be used to detect amplicons generated during target amplification. MNAzymes are formed when the target sensor arms of the partzymes each hybridize to a target or target amplicon through complementary base pairing, forming the active catalytic core of the MNAzyme. The loop region of the LOCS oligonucleotide hybridizes to the substrate binding arm of the MNAzyme through complementary base pairing, and the substrate within the loop is cleaved by the MNAzyme. This generates a split LOCS structure with a stem of Tm B, which is lower than Tm A of the intact LOCS. Tm Measurement of the fluorescent signal at a temperature above Tm B but below Tm A indicates the presence of the target in the reaction. One of skill in the art will recognize that the target can be detected in real time or at the end of the reaction.

[0291] Referring to the exemplary embodiment shown in Figure 3, a standard linear MNAzyme substrate is shown used in combination with a single LOCS probe containing an MNAzyme substrate within its loop. Both the linear MNAzyme substrate and the single LOCS probe may be labeled with the same (or similar) detection moiety, e.g., a specific fluorophore (F) / quencher (Q) dye pair. Alternative detection moieties described herein may also be used. The linear substrate contains a first substrate sequence cleavable by a first MNAzyme that assembles in the presence of a first target (Figure 3A). In the presence of the first target, the linear substrate is cleaved by the first MNAzyme, resulting in an increase in fluorescence that can be detected over a wide range of temperatures. The LOCS contains a second substrate sequence cleavable by a second MNAzyme that assembles in the presence of a second target (Figure 3B). In the presence of the second target, the LOCS is cleaved to generate a split LOCS that melts at a melting temperature (Tm B) lower than the melting temperature (Tm A) of the intact LOCS. At temperatures below Tm B, the stem portion of the split-LOCS remains hybridized, and thus the fluorophore is quenched due to its proximity to the quencher molecule. At temperatures above Tm B, the stem portion of the split-LOCS dissociates, separating the fluorophore from the quencher molecule and resulting in an increase in fluorescence. If both targets are present and fluorescence is measured at a first temperature below Tm B, the increase in fluorescence is associated with only the first target 1. If fluorescence is measured at a second temperature above Tm B but below TmA, the increase in fluorescence is associated with the first target and / or the second target. If both target 1 and target 2 are present, the observed change in fluorescence during amplification at the second temperature will be greater than the change at the first temperature, thus allowing for the determination of whether target 1, target 2, or targets 1 and 2 are present in the reaction, or whether neither target is present in the reaction.

[0292] In other embodiments of the invention, alternative LOCS structures useful in combination with standard reporter probes can be used. As illustrated in Figures 4A and 4B, the loop region of the LOCS oligonucleotide can contain a target-specific sequence that is fully or partially complementary to the target to be detected and, if double-stranded, can serve as a substrate (Figure 3A) for degradation by an exonuclease, e.g., by the inherent exonuclease activity of a polymerase. In yet another embodiment, shown in Figure 3B, the target-specific sequence within the loop can further comprise one strand of a double-stranded restriction enzyme recognition site. Hybridization of the loop sequence to the target sequence can result in a functional, cleavable restriction site. In a preferred embodiment, the restriction enzyme is a nicking enzyme that can cleave the loop strand of the LOCS oligonucleotide while leaving the target intact.

[0293] In various embodiments of the invention, different combinations of known reporter probes or substrates can be combined with a LOCS probe in a single reaction. As a non-limiting example, a reaction for detection of two targets may include any combination of a first probe selected from Group 1, including but not limited to, a linear MNAzyme substrate, a TaqMan or hydrolysis probe, a molecular beacon, a sloppy beacon, an eclipse probe, a Scorpion uniprobe, a Scorpion biprobe, a capture / pitcher oligonucleotide, and a dual hybridization probe, and a second probe selected from Group 2, including but not limited to, a LOCS probe comprising a universal MNAzyme substrate, a LOCS probe comprising a target-specific substrate for an exonuclease, and a LOCS probe comprising a target-specific substrate for an endonuclease such as a nicking enzyme.

[0294] Any combination of Group 1 probes and Group 2 probes can be used to measure multiple targets in a single reaction according to the methods of the present invention. The embodiment shown in Figure 3 illustrates an exemplary combination of a linear MNAzyme substrate cleavable by a first MNAzyme and a LOCS probe cleavable by a second MNAzyme. Another non-limiting embodiment of the present invention is shown in Figure 5, in which a non-cleavable molecular beacon may be combined with a LOCS probe cleavable by an MNAzyme. Both the molecular beacon and the LOCS probe may be labeled with the same (or similar) detection moiety, e.g., the same fluorophore or a fluorophore emitting at a similar wavelength. The molecular beacon may have a stem region with Tm A and a loop region capable of specifically hybridizing to a first target 1 with Tm B, where Tm B is higher than Tm A. This can be combined with an intact LOCS probe which may have a stem region with a Tm C and a loop region which can be cleaved by an MNAzyme in the presence of a second target 2, thus generating a split LOCS with a Tm D, which is less than Tm C. The presence of target 1 and / or target 2 can be distinguished by measuring fluorescence at two temperatures in real time, or by using separate measurements taken at or near the start of amplification and after amplification.

[0295] - LOCS combinations including reversible probes In some embodiments, the compositions and methods of the present invention comprise a combination of a LOCS and an oligonucleotide probe capable of generating a target-dependent detectable signal that can be reversibly modulated by temperature. Additionally, the LOCS and oligonucleotide probe may be amenable to modulation of target-independent signal generation by temperature, thereby allowing manipulation of background noise or baseline levels.

[0296] For example, an oligonucleotide probe may adopt a first conformation or configuration in the absence of a target that inhibits emission of a detectable signal, and a second conformation or configuration in the presence of a target that promotes emission of a detectable signal indicative of the presence of the target. Non-limiting examples of oligonucleotide probes in this category include molecular beacons, sloppy beacons, Scorpion uniprobes, Scorpion biprobes, and capture / pitcher oligonucleotides.

[0297] Alternatively, in the case of a dual hybridization probe, two additional oligonucleotides in addition to the LOCS may be arranged in such a way that a detectable signal is suppressed in the presence of the target and a detectable signal is generated in the absence of the target.

[0298] In the above embodiments, the intact LOCS undergoes a target-dependent cleavage event to provide a split LOCS, and the double-stranded stem portion of the split LOCS can be designed to dissociate at a temperature different from the temperature at which a target-dependent change in conformation or configuration of the first oligonucleotide and associated detectable signal is produced.

[0299] In some embodiments, the oligonucleotide is a molecular beacon. The following scenarios provide non-limiting examples of multiplex detection assays according to embodiments of the present invention. In these scenarios, a molecular beacon can be used in combination with a LOCS for the detection of target 1 and target 2, respectively. The molecular beacon may have a melting temperature of its double-stranded stem portion, Tm A, and a melting temperature of the duplex formed between its single-stranded loop duplex and target 1, Tm B. The LOCS may have a melting temperature of its double-stranded stem portion when intact, Tm C, and a melting temperature of its double-stranded stem portion when cleaved, Tm D. The opposing strand of the double-stranded stem portion of the molecular beacon may be labeled with a fluorophore and a quencher, similar to those of the LOCS. The fluorophores of the molecular beacon may be the same or may emit in the same region of the visible spectrum as the fluorophores of the LOCS. In alternative embodiments, different detection moieties may be utilized, including, for example, nanoparticles of the same or similar size and / or type for colorimetric or SPR detection, reactive moieties (e.g., alkaline phosphatase or peroxidase enzymes) for chemiluminescent detection, electroactive species (e.g., ferrocene, methylene blue, or peroxidase enzymes) for electrochemical detection. Those skilled in the art will readily appreciate that the molecular beacons in these cases may be replaced with Sloppy Beacons, Scorpion Uniprobes, or Scorpion Biprobes.

[0300] Various relationships may exist between the temperature at which the reaction is measured and the melting temperatures of various regions of the reporter probe. Four scenarios are described in detail below within the context of a reaction involving one molecular beacon and one LOCS probe, and non-limiting exemplary temperatures for such scenarios are outlined in Table 1 below.

[0301] [Table 1]

[0302] Scenario 1 In a first non-limiting example, Tm A may be higher than Tm D, and Tm B may be higher than Tm The presence of target 1 and / or target 2 can be identified by measuring fluorescence at two temperatures, taken at or near the start of amplification and after amplification. In the presence of target 1 and / or target 2, measuring fluorescence at a first temperature 1, which may be below Tm A, Tm B, and Tm D, can generate a signal indicative of the presence of target 1 only. At this temperature, the molecular beacon hybridizes to target 1 (if present) and fluoresces, whereas in the absence of target 1, its stem remains internally hybridized and is therefore quenched. At temperature 1, both intact and / or split LOCS species are quenched due to hybridization of their respective stems at this temperature. Furthermore, in the presence of target 1 and / or target 2, measuring fluorescence at a second temperature 2, which is higher than both temperature 1 and Tm D but lower than both Tm B and Tm C, can indicate the presence of target 1 and / or target 2. At this second temperature, the molecular beacon hybridizes to target 1 (if present) and fluoresces, whereas in the absence of target 1, its stem remains internally hybridized and therefore quenched. Furthermore, at this second temperature, in the absence of target 2, the LOCS probe remains intact and quenched, but is cleaved by the target 2-specific MNAzyme (if present), causing its stem to dissociate and produce fluorescence. In this scenario, if fluorescence at temperature 1 increases during amplification, this indicates the presence of target 1. Furthermore, if the increase in fluorescence observed at temperature 2 over the course of amplification is greater than that observed at temperature 1, this indicates the presence of target 2.

[0303] Scenario 2 In a second non-limiting example, Tm A may be similar to Tm D, and Tm B may be similar to Tm The Tm B may be similar to Tm C, and Tm B may be higher than Tm D. The presence of target 1 and / or target 2 can be identified by measuring fluorescence at two temperatures in real time, or by using single measurements taken at or near the start of amplification and after amplification. In the presence of target 1 and / or target 2, measuring fluorescence at a first temperature 1, which may be below Tm A, below Tm B, and below Tm D, can generate a signal indicating the presence of target 1 only. At this temperature, the molecular beacon hybridizes to target 1 (if present) and fluoresces, while its stem remains internally hybridized and quenched in the absence of target 1. At this first temperature 1, both intact and / or split LOCS species are quenched due to hybridization of the stem region at this temperature. Furthermore, in the presence of target 1 and / or target 2, measuring fluorescence at a second temperature 2, which is higher than both Tm A and Tm D but lower than both Tm B and Tm C, can indicate the presence of target 2. At this second temperature, the molecular beacon hybridizes to target 1 (if present) and fluoresces, or fluoresces target-independently due to the dissociation and opening of its stem at this temperature. Therefore, the molecular beacon fluoresces regardless of the presence or absence of any target, giving a background fluorescence level at this temperature that may not change during amplification. Furthermore, at this second temperature, in the absence of target 2, the LOCS probe remains intact and quenched, but is cleaved by the target 2-specific MNAzyme (if present), causing its stem to dissociate and thus generate fluorescence. In this scenario, if fluorescence at temperature 1 increases during amplification, this indicates that target 1 is present and detected by the molecular beacon, and if fluorescence at temperature 2 during amplification indicates that target 2 is present and detected by the LOCS probe. Qualitative data can be obtained using discrete temperature measurements at / near the beginning and end of PCR, and / or quantitative data can be read directly from the two amplification curves generated at each temperature during PCR.Alternatively, if Tm A and Tm D are dissimilar, the same relationship between fluorescence levels at the two temperatures and the presence or absence of target will hold if both Tm A and Tm D are above temperature 1 and below temperature 2.

[0304] The above approach may offer significant advantages over other methods known in the art that utilize measurements at multiple temperatures to distinguish multiple targets at a single wavelength, such as TOCE. TOCE measures fluorescence from a first target at a first temperature and from two targets (first target + second target) at a second temperature. This data is analyzed to mathematically subtract the amount of fluorescence associated with the first target at the second temperature to quantify the second target in a complex analysis, which further requires adjustments to account for inherent differences in fluorescence associated with temperature itself. Embodiments of the invention described herein utilize molecular beacons and LOCS probes in a manner that eliminates the need for complex post-PCR analysis, enabling direct quantification of a first target from a first amplification curve generated at a first temperature and a second target from a second amplification curve generated at a second temperature. These embodiments measure each target individually, and furthermore, because each target only produces a signal detectable above background at one of the two temperatures selected for data acquisition, no adjustments are required to account for differences in the fluorescence output of the same molecule.

[0305] Scenario 3 In a third non-limiting example, Tm A may be lower than Tm D, and Tm B may be lower than Tm The Tm C may be similar to Tm D, and Tm C may be higher than Tm B. The presence of target 1 and / or target 2 can be identified by measuring fluorescence at two temperatures in real time, or by using single measurements taken at or near the beginning of amplification and after amplification. In the presence of target 1 and / or target 2, measuring fluorescence at a first temperature 1, which may be below Tm A, below Tm B, and below Tm D, can generate a signal indicative of the presence of target 1 only. At this temperature, the molecular beacon hybridizes to target 1 (if present) and fluoresces, while its stem remains internally hybridized and quenched in the absence of target 1. At this temperature 1, both intact and / or split LOCS species are quenched due to hybridization of their stems at this temperature. Furthermore, in the presence of target 1 and / or target 2, at temperatures 1 and Tm D and Tm Measurement of fluorescence at a second temperature 2, higher than both Tm A and Tm B but lower than Tm C, can indicate the presence of target 2. At this second temperature, the molecular beacon cannot hybridize to target 1 and always has an open, dissociated stem, and therefore fluoresces regardless of the presence or absence of any target, giving a background fluorescence level at this temperature that may not change during amplification. Furthermore, at this second temperature, in the absence of target 2, the LOCS probe remains intact and quenched, but is cleaved by the target 2-specific MNAzyme (if present), causing its stem to dissociate and thus generate fluorescence. In this scenario, if fluorescence at temperature 1 increases during amplification, this indicates that target 1 is present and detected by the molecular beacon, and an increase in fluorescence at temperature 2 indicates that target 2 is present and detected by the LOCS probe. Qualitative data can be obtained using discrete temperature measurements at / near the beginning and end of PCR, and / or quantitative data can be read directly from the two amplification curves generated at each temperature during PCR. Alternatively, if Tm A and Tm D are similar, or if Tm A is higher than Tm D, then if both Tm A and Tm D are higher than Temperature 1 and lower than Temperature 2, the same relationship between fluorescence levels at the two temperatures and the presence or absence of target will hold.

[0306] Scenario 4 In a fourth non-limiting example, both Tm A and Tm B may be higher than Tm C and Tm D. The presence of target 1 and / or target 2 can be identified by measuring fluorescence at two temperatures taken at or near the beginning of amplification and after amplification. In the presence of target 1 and / or target 2, measuring fluorescence at a first temperature 1, which may be below Tm A and Tm B but above Tm C and Tm D, may generate a signal indicative of the presence of target 1. At this temperature, the molecular beacon will hybridize to target 1 (if present). At this second temperature, the LOCS will fluoresce and fluoresce, while its stem remains internally hybridized in the absence of target 1 and is quenched. At this temperature, the LOCS fluoresces regardless of the presence or absence of target 2, and therefore contributes only to background, which does not change during amplification. In the absence of target 2, the stem of the intact LOCS dissociates and fluoresces; similarly, in the presence of target 2, the stem of the split LOCS dissociates and fluoresces. Furthermore, an increase in fluorescence during the amplification process at temperature 2, which is below temperature 1 and below Tm C, Tm A, and Tm B, but above Tm D, can indicate the presence of target 1 and / or target 2. At this second temperature, the molecular beacon will hybridize to target 1 (if present) and therefore fluoresce, or will remain quenched by the hybridized stem in the absence of target 1. Furthermore, at this second temperature, if target 2 is absent, the intact LOCS probe stem remains hybridized and quenched, or if target 2 is present, the LOCS is split by an MNAzyme specific for target 2, and the split LOCS stems dissociate and fluoresce. In this scenario, if fluorescence at temperature 2 increases during amplification, this indicates the presence of target 1 and / or target 2. Furthermore, if the increase in fluorescence observed over the course of amplification at temperature 2 is greater than that observed at temperature 1, this indicates the presence of target 2.

[0307] - LOCS combination including catcher-pitcher probes In some embodiments, the compositions and methods of the present invention may include a combination of a first oligonucleotide that functions as a catcher component of the LOCS and TOCE assays. This combination may enable simultaneous detection and quantification of two targets in a single fluorescent channel, for example, by acquiring fluorescent readings at two temperatures in real time during PCR. Alternatively, in the absence of real-time monitoring, the approach may be applied to fluorescent data collected, for example, at or near the start of amplification and at discrete time points after amplification.

[0308] In one embodiment, a first oligonucleotide containing a catcher can be combined with a LOCS probe, both of which can be labeled with the same fluorophore and quencher moieties for simultaneous detection in the same fluorescence channel. The reaction can also include a pitcher containing a single-stranded oligonucleotide containing a 5' tag region complementary to the catcher and a 3' sensor region complementary to the first target 1. The catcher can include a single-stranded oligonucleotide labeled with a quencher at the 5' end, a fluorophore downstream of the quencher, and a 3' region complementary to the tag portion of the pitcher. When the catcher is in a single-stranded conformation, the fluorophore is in close proximity to the quencher and the signal is quenched.

[0309] In a non-limiting example of an amplification reaction such as PCR, the primer and 3' sensor region of a pitcher may hybridize to target 1. During primer extension using target 1 or its amplicon as a template, the pitcher may be degraded by the exonuclease activity of a DNA polymerase, resulting in the release of the tag portion. The released tag may then hybridize to the complementary 3' portion of a catcher and be extended by a DNA polymerase, thus generating a double-stranded catcher duplex with Tm A, in which the fluorophore and quencher are separated, resulting in increased fluorescence indicating the presence of target 1. Furthermore, the reaction may contain an intact LOCS probe with stem and loop regions with Tm C, which may be cleaved by an MNAzyme in the presence of a second target 2, to generate a split LOCS with Tm D, which is lower than Tm A. Various relationships may exist between the temperature at which the reaction is measured and the melting temperatures of the catcher duplex and the LOCS reporter. Three scenarios are described in detail below within the context of reactions involving one Catcher probe and one LOCS probe, and non-limiting exemplary temperatures for such scenarios are outlined in Table 2 below.

[0310] [Table 2]

[0311] In a first non-limiting scenario (see Table 2), if the signal is measured at a first detection temperature below Tm A, Tm C, and Tm D in the presence of Target 1, the Catcher duplex may form and fluoresce, whereas in the absence of Target 1, the single-stranded Catcher remains quenched. At this temperature, both the intact and split LOCS stems are hybridized and therefore quenched regardless of the presence or absence of Target 2. Thus, an increase in fluorescence during amplification indicates Target 1. Furthermore, fluorescence measurements at a second detection temperature above Tm D but below Tm A and Tm C can indicate the presence of Target 1 and / or Target 2. At this second temperature, the Catcher remains single-stranded and quenched in the absence of target, and forms a duplex and fluoresces in the presence of target. Furthermore, at this second temperature, in the absence of target 2, the LOCS probe remains intact and quenched, but in the presence of target 2, it is cleaved, causing its stem to dissociate and produce fluorescence. In this scenario, if fluorescence at temperature 1 increases during amplification, this indicates the presence of target 1. Furthermore, if the increase in fluorescence observed at temperature 2 over the course of amplification is greater than that observed at temperature 1, this indicates the presence of target 2. An increase in fluorescence at the second temperature but not at the first temperature indicates the presence of only target 2.

[0312] In a second, non-limiting scenario (see Table 2), specific detection of a first target at a first detection temperature can be achieved according to Scenario 1 above. Furthermore, at a second detection temperature higher than Tm A and Tm D but lower than Tm C, the temperature is higher than the temperature at which the catcher duplex can form, and therefore the catcher duplex is dissociated (i.e., single-stranded) and quenched, regardless of the presence or absence of Target 1 and / or Target 2. In the absence of Target 2, all LOCSs are intact and quenched, but in the presence of Target 2, split LOCSs are generated, their stems dissociate, and an increase in fluorescence can be observed. Thus, an increase in fluorescence during PCR at the first temperature indicates the presence of Target 1, regardless of the presence or absence of Target 2, and conversely, an increase in fluorescence during PCR at the second temperature indicates the presence of Target 2, regardless of the presence or absence of Target 1. Thus, the combination of LOCS and Catcher-Pitcher probes may allow detection of target 1 only using the Catcher-Pitcher probe, as monitored by an increase in fluorescence above background at a first temperature, and may allow detection of target 2 only using the LOCS probe, as monitored by an increase in fluorescence above background at a second temperature.

[0313] In a third non-limiting scenario (see Table 2), if the signal is measured in the presence of target 1 alone at a first detection temperature below Tm A but above Tm C and Tm D, the catcher duplex may form and fluoresce, whereas in the absence of target 1, the single-stranded catcher remains quenched. At this temperature, both the intact and split LOCS stems dissociate, generating high levels of background fluorescence, regardless of the presence or absence of target 2. Thus, an increase in fluorescence above background fluorescence during amplification indicates target 1 alone. Furthermore, fluorescence measurements at a second detection temperature below Tm A and Tm C but above Tm D may indicate the presence of target 2 alone. At this second temperature, the catcher remains single-stranded and quenched in the absence of target, and forms a duplex and fluoresces in the presence of target. Furthermore, at this second temperature, the LOCS probe remains intact and quenched in the absence of target 2, but is cleaved in the presence of target 2, causing its stem to dissociate and generate fluorescence. In this scenario, if fluorescence at temperature 1 increases during amplification, this indicates the presence of target 1 only, and if fluorescence at temperature 2 increases during amplification, this indicates the presence of target 2 only.

[0314] In another non-limiting format, for example, for detection by SPR, the catcher can be bound to a gold nanoparticle (GNP) and free in solution, while the pitcher can be bound to a gold surface. In the presence of target 1 and at a temperature below Tm A, a catcher bilayer forms, and the GNP The catcher can be brought into proximity with the gold surface, generating a measurable shift in the SPR signal. However, in the absence of target 1, the catcher is single-stranded and free in solution (i.e., not in proximity to the gold surface), and therefore does not generate any measurable shift in the SPR signal above the baseline SPR signal. Therefore, any measurable shift in the SPR signal at this temperature indicates the presence of target 1 in the sample. Furthermore, the LOCS may be bound to a GNP at one end and to the gold surface at the other end. In the absence of target 2, the GNP is always bound, whereas in the presence of target 2, a split LOCS is generated; therefore, in this case, the GNP is in proximity to the gold surface only when the detection temperature is below the temperature of the split LOCS. As mentioned above, in a scenario similar to Scenario 2 above, if the first detection temperature is below Tm B, Tm C, and Tm D, a change in signal indicates the presence of target 1, as the GNP on the catcher is in proximity to the gold surface. If the second detection temperature is below Tm C but above Tm A and Tm D, a change in signal indicates the presence of target 2 as the GNPs on the split LOCS detach from the gold surface.

[0315] In yet another format, for example, colorimetric detection, both the catcher and the LOCS probe may be labeled with GNPs at both ends. Then, at a first temperature below Tm A, Tm C, and Tm D, the presence of target 1 results in a measurable color change from purple (aggregated GNPs) to red (dispersed GNPs), regardless of the presence or absence of target 2. Then, at a second temperature above Tm A and Tm D but below Tm C, the presence of target 2 results in a measurable color change from purple (aggregated GNPs) to red (dispersed GNPs), regardless of the presence or absence of target 1.

[0316] In yet another format, the catcher can be labeled with an electroactive moiety, such as methylene blue or ferrocene, and the catcher can be bound to the electrode surface. At a first temperature (below Tm A), the catcher duplex forms on the electrode surface (if target 1 is present), bringing the electroactive moiety into close proximity to the electrode surface and generating a measurable shift in the electrochemical signal (i.e., oxidation or reduction current). However, in the absence of target 1, the catcher is free in solution and not in close proximity to the electrode surface, and therefore does not generate any measurable shift in the electrochemical signal (i.e., oxidation or reduction current) above the baseline signal. Therefore, any measurable shift in the electrochemical signal at this temperature indicates the presence of target 1 in the sample.

[0317] -LOCS combined with dual hybridization probes In some embodiments, the compositions and methods of the present invention comprise a combination of a LOCS and an oligonucleotide probe comprising two target-specific components.

[0318] A dual hybridization probe may contain a first oligonucleotide having a Tm A and a second oligonucleotide having a Tm B, where Tm A and Tm B may be equal or different. The first oligonucleotide may be labeled with a fluorophore at its 3' end, and the second oligonucleotide may be labeled with a quencher at its 5' end. (Alternatively, one oligonucleotide may be labeled with a quencher at its 3' end and the other with a fluorophore at its 5' end.) The two oligonucleotide probes hybridize adjacently or substantially adjacently (e.g., with less than a 2-, 3-, 4-, or 5-nucleotide gap) on target 1, forming a duplex structure with a Tm equal to the lower of Tm A and Tm B. In this scenario, a preferred first temperature for data acquisition is one where Tm A and Tm B are equal. The first temperature may be less than Tm B and less than Tm C and Tm D, i.e., the Tms of the stems of the intact LOCS and split LOCS, respectively. At this first temperature, the first and second oligonucleotides are free in pre-amplification solution and fluorescent in the absence of Target 1, but hybridization to Target 1, if present, brings the fluorophore and quencher into close proximity. Furthermore, at the first temperature, both the intact and split LOCS stems are quenched. Thus, the decrease in fluorescence observed at the first temperature indicates the presence of Target 1, regardless of the presence or absence of Target 2.

[0319] The LOCS probe may be cleaved by the MNAzyme in the presence of a second target 2, and data acquisition may be performed at a second temperature above Tm A and / or Tm B, above Tm D but below Tm C. In this scenario, the intact LOCS remains quenched before amplification and in the absence of target, but in the presence of target 2, the split LOCS stem dissociates, resulting in an increase in fluorescence. At this temperature, the first and second oligonucleotides fluoresce regardless of the presence or absence of target 1, as they cannot hybridize to target 1 at this temperature. This fluorescence contributes to the background signal at this temperature. Thus, an increase in fluorescence above background after amplification indicates the presence of target 2, regardless of the presence or absence of target 1.

[0320] This combination therefore allows for detection of target 1 using the dual hybridization probe, determined as a decrease in fluorescence at a first temperature, and detection of only target 2 using the LOCS probe, determined by an increase in fluorescence at a second temperature.

[0321] In another format, for example, detection by SPR, a first oligonucleotide can be bound to a gold nanoparticle (GNP), and a second oligonucleotide can be bound to a gold surface. At a first temperature, the two oligonucleotide probes can hybridize adjacently on target 1 (if present), forming a duplex structure and bringing the GNP into close proximity to the gold surface, which can generate a measurable shift in the SPR signal. However, in the absence of target 1, the first oligonucleotide can be free in solution and not in close proximity to the gold surface, and therefore will not generate any measurable shift in the SPR signal above the baseline SPR signal. Therefore, any measurable shift in the SPR signal at this temperature can indicate the presence of target 1 in the sample.

[0322] In yet another format, for example, colorimetric detection, both the first and second oligonucleotides may be labeled with gold nanoparticles. At a first temperature, the first and second oligonucleotides may be free in solution and exhibit a red color in the absence of target 1. However, in the presence of target 1, the two oligonucleotide probes may hybridize adjacently on the target, forming a duplex structure, bringing the GNPs into close proximity with each other and generating a measurable color change from red to purple. Thus, the measurable color change from red to purple may indicate the presence of target 1 in the sample.

[0323] In yet another format, a first oligonucleotide can be labeled with an electroactive moiety such as methylene blue or ferrocene, and a second oligonucleotide can be attached to the electrode surface. At a first temperature, the two oligonucleotide probes (if present) hybridize adjacently on target 1, forming a duplex structure on the electrode surface, bringing the electroactive moiety into close proximity to the electrode surface and generating a measurable shift in the electrochemical signal (i.e., oxidation or reduction current). However, in the absence of target 1, the first oligonucleotide can be free in solution and not in close proximity to the electrode surface, and therefore will not generate any measurable shift in the electrochemical signal (i.e., oxidation or reduction current) above the baseline signal. Therefore, any measurable shift in the electrochemical signal at this temperature can indicate the presence of target 1 in the sample.

[0324] - LOCS combinations containing digestible probes In some embodiments, the compositions and methods of the present invention involve LOCS and oligonucleotide probes that generate target-dependent detectable signals that cannot be reversibly modulated by temperature. However, LOCS remains amenable to modulation of signal generation by temperature in a target-independent manner, thus allowing manipulation of background noise or baseline levels.

[0325] For example, a first oligonucleotide probe for a first target 1 may undergo a target-dependent modification that provides a detectable signal that cannot be suppressed when the detection temperature is changed. The target-dependent modification may be cleavage or digestion of the first oligonucleotide, thereby triggering a detectable signal indicating the presence of the target. Non-limiting examples of oligonucleotide probes in this category include TaqMan probes, MNAzyme substrates, and probes that can be cleaved by restriction enzymes in a target-dependent manner. After modification, the signal generated by these probes can be measured over a wide range of temperatures.

[0326] In these embodiments, the intact LOCS designed to detect the second target 2 can undergo cleavage to generate a split LOCS only in the presence of this target. Measurement of target 2 requires detection at a temperature below the Tm of the stem of the intact LOCS but above the Tm of the stem of the split LOCS. In contrast, the temperature at which the first target is detected can be either below the Tm of both the intact and split LOCS stems or above the Tm of both the intact and split LOCS stems. If the first detection temperature is below the Tm of the stem, the background signal is suppressed because both the intact and / or split stems remain associated and quenched, whereas if the first detection temperature is above the Tm of the stem, the background signal becomes higher due to dissociation of both the intact and / or split stems. Thus, detection of a signal at the first temperature can be specific for target 1, regardless of the presence or absence of target 2. Detection of a signal at the second temperature indicates the presence of target 1 and / or target 2; if the increase in signal observed at the second temperature is greater than that observed at the first temperature, this indicates the presence of target 2. If a change in signal is observed only at the second temperature, but not at the first temperature, this indicates the presence of only the second target.

[0327] The following scenarios provide non-limiting examples of multiplex detection assays according to embodiments of the present invention and are summarized in Table 3 below. In these scenarios, the LOCS may have a melting temperature of its double-stranded stem portion when intact, Tm A, and a melting temperature of its double-stranded stem portion when cleaved, Tm B. The oligonucleotide probe (e.g., an MNAzyme substrate), as well as the opposing strand of the double-stranded stem portion of the LOCS, may be labeled with a fluorophore and a quencher. The fluorophore of the oligonucleotide probe may be the same or may emit in the same region of the visible spectrum as the fluorophore of the LOCS. In alternative embodiments, different detection moieties may be utilized, including, for example, nanoparticles of the same or similar size and / or type for colorimetric or SPR detection, reactive moieties (e.g., alkaline phosphatase or peroxidase enzymes) for chemiluminescent detection, or electroactive species (e.g., ferrocene, methylene blue, or peroxidase enzymes) for electrochemical detection. Those skilled in the art will readily appreciate that the oligonucleotide probe can be, for example, an MNAzyme substrate, a TaqMan probe, a hydrolysis probe, or a target-dependent restriction enzyme cleavable probe (RE probe).

[0328] The change in signal due to the presence or absence of a first target can be obtained by measuring the fluorescence after amplification (post-PCR) at a first temperature normalized to background fluorescence obtained at or near the start of amplification to PCR (pre-PCR) at that same first temperature, while the change in signal due to the presence or absence of a second target can be obtained by taking the total post-PCR fluorescence at a second temperature normalized to background fluorescence measured pre-PCR at that second temperature. Alternatively, the change in signal due to the presence or absence of the first and second targets can be obtained by measuring the total post-PCR fluorescence at that second temperature normalized to background fluorescence measured pre-PCR at that second temperature. The change in signal due to the presence or absence of each can be obtained by measuring the total fluorescence after amplification at the first and second temperatures, both of which can be normalized to background fluorescence at a third temperature taken before PCR of the same reaction or at any time in an equivalent control reaction known to lack the target. A background measurement can be made at a third temperature if the first temperature is less than the second temperature and the third temperature is less than the Tm of the intact stem.

[0329] In all scenarios, the presence or absence of a signal indicative of the first target is measured at a first temperature using a first probe, which can be, for example, an MNAzyme substrate, a TaqMan probe, or an RE probe. Prior to amplification, this probe is quenched, but if target 1 is present, the probe is cleaved during amplification, resulting in an increase in fluorescence that is detectable at both the first and second temperatures. The presence or absence of the second target is determined at a second temperature using a LOCS probe, which is always above the Tm of the split LOCS (Tm B) but below the Tm of the intact LOCS (Tm A).

[0330] [Table 3]

[0331] Scenario 1. In a first non-limiting example, the first temperature is lower than the second temperature and lower than Tm A and Tm B, as set forth in Table 3. An increase in fluorescence at the first temperature indicates the presence of Target 1, and the background signal reflects the presence of quenched LOCS, with both intact and / or split stems remaining associated. The background at the first and second temperatures may be measured prior to PCR, respectively, at each temperature. Alternatively, a background measurement may be measured at a third temperature, which is lower than both the first and second temperatures.

[0332] Scenario 2 In a second non-limiting example, the first temperature is higher than the second temperature and higher than Tm A and Tm B, as set forth in Table 3. An increase in fluorescence at the first temperature indicates the presence of Target 1, and the background signal reflects fluorescence from intact and / or split LOCS with dissociated stems. The background at the first and second temperatures, respectively, may be measured prior to PCR at each temperature.

[0333] Scenario 3 In a third non-limiting example, the first temperature is lower than the second temperature and lower than Tm A and Tm B, as set forth in Table 3. An increase in fluorescence at the first temperature indicates the presence of Target 1, and the background signal reflects the presence of quenched LOCS, with both intact and / or split stems remaining associated. The background at the first and second temperatures may be measured prior to PCR, respectively, at each temperature. Alternatively, a background measurement may be measured at a third temperature, which is lower than the second temperature but higher than the first temperature.

[0334] In other formats, such as SPR detection, the first oligonucleotide and LOCS probe can be attached to a gold nanoparticle (GNP) at one end and to a gold surface at the other end. In the absence of target, the first oligonucleotide can remain intact, and the GNP can be in close proximity to the gold surface, generating a baseline level SPR signal. In the presence of target 1, the first oligonucleotide can be cleaved, separating the GNP from the gold surface and generating a measurable shift in SPR signal, indicating the presence of the first target in the sample. This can be measured at a first temperature below Tm A and Tm B, so that GNPs bound to or associated with either intact or split LOCS can remain on the surface. Measuring a change in SPR signal at a second temperature above the Tm of the split LOCS can indicate the presence of the first and / or second target. Furthermore, if a change in signal is observed at the first temperature and a larger change is observed at the second temperature, this indicates the presence of target 2. Alternatively, if a signal is detected at the second temperature but not at the first temperature, this also indicates only the second target.

[0335] In another format, such as colorimetric detection, the first oligonucleotide probe and LOCS may be labeled with gold nanoparticles at both ends. At a first temperature below Tm A and Tm B, the first oligonucleotide may remain intact in the absence of target 1, and the GNPs may be in close proximity to each other and aggregate, exhibiting a purple color. However, in the presence of target 1, the first oligonucleotide may be cleaved, separating the GNPs and exhibiting a measurable color change from purple to red. Thus, the measurable color change from purple to red may indicate the presence of target 1 in the sample. At a second temperature above the first temperature, above Tm B but below Tm A, only in the presence of the second target may the LOCS be split, the GNPs are dispersed, and the color changes from purple to red. When both targets are present, the color shift may be more drastic.

[0336] In yet another format, the first oligonucleotide may be labeled with an electroactive moiety such as methylene blue or ferrocene at one end and attached to the electrode surface at the other end. In the absence of target 1, the first oligonucleotide may remain intact and the electroactive species may be in close proximity to the electrode surface, generating a baseline level electrochemical signal (i.e., oxidation or reduction current). However, in the presence of target 1, the first oligonucleotide may be cleaved and the electroactive moiety may be released into solution, generating a measurable shift in the electrochemical signal (i.e., oxidation or reduction current) above that of the baseline signal. Thus, any measurable shift in the electrochemical signal at this temperature may indicate the presence of target 1 in the sample.

[0337] Further exemplary embodiments In certain embodiments, reporter oligonucleotides, including LOCS oligonucleotides of the present invention, can be used to directly detect targets. In other exemplary embodiments, reporter probes or substrates can be used to detect target amplicons generated by target amplification techniques, including, but not limited to, PCR, RT-PCR, SDA, NEAR, HDA, RPA, LAMP, RCA, TMA, 3SR, LCR, RAM, or NASBA. Cleavage or degradation resulting in LOCS cleavage can occur in real time during target amplification or can be performed at the end point of the reaction following amplification. The loop region can be cleaved by target-dependent cleavage or degradation mediated by the enzymatic activity of catalytic nucleic acids, including, but not limited to, MNAzymes, DNAzymes, and ribozymes, or by the enzymatic activity of protein enzymes, including exonucleases or endonucleases. As a non-limiting example, exonuclease activity can be the intrinsic catalytic activity of, for example, a polymerase. As a non-limiting example, the endonuclease activity can be the intrinsic catalytic activity of a restriction enzyme, including, for example, a nicking endonuclease, a riboendonuclease, or a dual-specific nuclease (DSN).

[0338] The reactions of the present invention are designed to simultaneously detect multiple targets using a single LOCS oligonucleotide in combination with other types of reporter probes. As will be apparent to those skilled in the art, a standard reporter probe may be further combined with additional LOCSs, for example, each LOCS may contain a different universal substrate within its loop and a different stem region that can melt at different temperatures following cleavage of the substrate / loop by different MNAzymes.

[0339] The reaction mixture may further include an additional reporter probe or substrate combined with the LOCS labeled with a different fluorophore and quencher pair. As a non-limiting example, reporter oligonucleotide 1 and intact LOCS oligonucleotide 2 can be labeled with fluorophore A, and reporter oligonucleotide 3 and intact LOCS oligonucleotide 4 can be labeled with fluorophore B. In one embodiment, in which reporter oligonucleotides 1 and 3 are linear MNAzyme substrates, MNAzyme 1 can form in the presence of target 1 and cleave reporter oligonucleotide 1 to generate fluorescence at all temperatures, and MNAzyme 2 can form in the presence of target 2 and cleave substrate 2 within LOCS oligonucleotide 2 to yield cleaved split-LOCS structure 2 containing stem 2 that melts at temperature X. MNAzyme 3 can form in the presence of target 3 and cleave reporter oligonucleotide 3 to generate fluorescence at all temperatures. MNAzyme 4 can form in the presence of target 4 and cleave substrate 4 within LOCS oligonucleotide 4 to yield split-LOCS structure 4 containing stem 4 that melts at temperature Y.

[0340] When fluorescence is analyzed at a temperature below both X and Y, excitation at the wavelength of fluorophore A indicates the presence of target 1, and excitation at the wavelength of fluorophore B indicates the presence of target 3. When fluorescence is analyzed at a temperature above both X and Y but below the melting temperatures of both oligonucleotides 2 and 4 of the intact LOCS, excitation at the wavelength of fluorophore A indicates the presence of target 1 and / or target 3. Excitation at the wavelength of fluorophore B indicates the presence of target 2, and excitation at the wavelength of fluorophore B indicates the presence of target 3 and / or target 4.

[0341] When the melting profile of the reaction is analyzed at the excitation wavelength of fluorophore A, the presence of a peak at a first temperature X and the absence of a peak at a second temperature 2 above temperature X indicates that LOCS reporter 2 has been cleaved / cleaved by the MNAzyme in the presence of target 2. Alternatively, the absence of a peak at temperature X and the presence of a peak at temperature 2 indicates that LOCS reporter 2 has remained intact and has not been cleaved by the MNAzyme due to the absence of target 2 in the sample. When the melting profile of the reaction is analyzed at the excitation wavelength of fluorophore B, the presence of a peak at a third temperature Y and the absence of a peak at a fourth temperature 4 above temperature Y indicates cleavage of LOCS reporter 4 by the MNAzyme in the presence of target 4, while the absence of a peak at temperature Y and the presence of a peak at temperature 4 indicates that LOCS reporter 4 has remained intact and has not been cleaved by the MNAzyme due to the absence of target 4 in the sample. Thus, analysis at two wavelengths read on two channels of the instrument can be used as a confirmatory tool to detect and differentiate targets, provided that the presence of the remaining two targets is determined using other detection means, such as real-time detection. Those skilled in the art will recognize that the strategy can be extended to monitor cleavage of more than two targets at one particular wavelength, further increasing the number of fluorophores analyzed, up to a number dictated by the maximum capacity of the instrument available for distinguishing between individual wavelengths.

[0342] In a further exemplary embodiment, a linear MNAzyme substrate and a LOCS oligonucleotide may be combined, both containing the same fluorophore / quencher dye pair, with the substrate region specific for a DNAzyme or ribozyme, e.g., a DNAzyme or ribozyme that can be catalytically active only in the presence of a particular metal ion. It is known in the art that certain DNAzymes and ribozymes require a metal cation cofactor to enable catalytic activity. For example, some DNAzymes and ribozymes can be catalytically active only in the presence of, e.g., lead or mercury. Such metals may be present, for example, in environmental samples. A reaction may include a linear MNAzyme substrate, e.g., a mercury-dependent DNAzyme; the presence of mercury in the sample may result in cleavage of the linear MNAzyme substrate and generation of a fluorescent signal. Alternatively, the same reaction may include a LOCS reporter containing a loop containing a substrate for a lead-dependent DNAzyme; the presence of lead in the sample may result in cleavage of the LOCS at a temperature above the Tm of the cleaved LOCS and generation of a fluorescent signal. An increase in fluorescence at a first temperature below the Tm of the split-LOCS indicates the presence of mercury. An increase in fluorescence at a second temperature above the Tm of the split-LOCS but below the Tm of the intact LOCS indicates the presence of mercury and / or lead. If mercury is detected at the first temperature, a further increase in fluorescence at the second temperature indicates the presence of lead. This can be confirmed by cooling the reaction so that the stems of the cleaved split-LOCS structure reanneal, and then performing a melting curve analysis to determine the presence or absence of a peak indicative of the presence of the split-LOCS structure. Those skilled in the art will readily recognize that multiple probes cleavable in the presence of a particular metal cofactor can be combined in a single reaction and detected in real time or at the end of the reaction.

[0343] Non-limiting examples of target nucleic acids (i.e., polynucleotides) that can be detected using LOCS oligonucleotides in combination with other well-known probe types 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 RNA, ribosomal RNA, derivatives thereof, amplicons thereof, or any combination thereof (including mixed polymers of deoxyribonucleotide and ribonucleotide bases).

[0344] Generation of a detectable signal The methods and compositions of the present invention utilize detection moieties to provide a detectable signal. The nature of the detectable signal a moiety is capable of producing depends on the type of detection moiety and / or the conformation of the oligonucleotide to which it is associated.

[0345] Any suitable detectable moiety can be utilized that is capable of providing a detectable signal upon modification of the oligonucleotide to which it is associated. Non-limiting examples of suitable detectable moieties include fluorophores for generating fluorescent signals, nanoparticles for generating colorimetric or SPR signals, reactive moieties for generating chemiluminescent signals (e.g., alkaline phosphatase or peroxidase enzymes), electroactive species for generating electrochemical signals, and any combination thereof. Non-limiting examples of suitable electroactive species include methylene blue, toluene blue, oracetone blue, ferrocene, Hoechst 33258, [Ru(phen)3]2+, or daunomycin. Common electrode materials include gold, glassy carbon, pencil graphite, or carbon ionic liquids. Methods for detecting and measuring fluorescent, chemiluminescent, colorimetric, surface plasmon resonance (SPR), and electrochemical signals are well known to those skilled in the art.

[0346] As a non-limiting example, an oligonucleotide of the present invention comprising a LOCS may have one or more fluorophores attached. The detectable signal originally generated by the fluorophore may be quenched due to proximity to one or more quencher molecules. For example, but not limited to, a fluorophore can be attached to one strand of the double-stranded stem portion (e.g., the 5' or 3' end) of a molecular beacon or LOCS, and a quencher can be attached to the opposite strand of the double-stranded stem portion (e.g., the 5' or 3' end). Alternatively, the quencher can be attached to another entity (e.g., a surface or another oligonucleotide) to which the oligonucleotide is attached, so that the detectable signal originally generated by the fluorophore can be quenched. In the presence of a target, the oligonucleotide may undergo a modification that distances the fluorophore from the quencher molecule, thereby generating a detectable signal.

[0347] Additionally or alternatively, oligonucleotides (including LOCS) can be attached to GNPs for colorimetric detection. When gold nanoparticles are aggregated in close proximity to each other, they exhibit a purple color (i.e., absorbance at longer wavelengths); when gold nanoparticles are separated, they exhibit a red color (i.e., absorbance at shorter wavelengths); a measurable color change from purple to red (e.g., LOCS, linear MNAzyme substrates, Catcher-Pitcher probes, TaqMan probes, and restriction enzyme probes), or alternatively, from red to purple (e.g., dual hybridization probes), indicates the presence of a specific target in a sample.

[0348] Additionally or alternatively, oligonucleotides (including LOCS) and / or oligonucleotide components may be attached to GNPs and / or gold surfaces for SPR detection of targets in a sample. When GNPs move into proximity, or alternatively, when they move away from the gold surface, they can generate a measurable change in SPR signal; a decrease in SPR signal using some approaches (e.g., LOCS, linear MNAzyme substrates, TaqMan probes, and restriction enzyme probes) can indicate the presence of a specific target in the sample, or alternatively, an increase in SPR signal using other approaches (e.g., catcher-pitcher probes and dual hybridization probes) can indicate the presence of a specific target in the sample.

[0349] Additionally or alternatively, oligonucleotide reporters and probes (including LOCS) and / or oligonucleotide components can be bound to electroactive species and / or electrode surfaces for electrochemical detection. When oligonucleotides bound to electroactive species migrate closer to the electrode surface, or alternatively, when they migrate away from the electrode surface, they can generate a measurable change in oxidation or reduction current. In some embodiments (e.g., LOCS, linear MNAzyme substrates, TaqMan probes, and restriction enzyme probes), the resulting measurable signal resulting from the electroactive species migrating away from the electrode surface indicates the presence of a specific target in the sample. Alternatively, in other embodiments (e.g., catcher-pitcher probes and dual hybridization probes), the resulting measurable signal resulting from the electroactive species migrating closer to the electrode surface indicates the presence of a specific target in the sample.

[0350] In some embodiments, the compositions and methods of the present invention utilize a LOCS conjugated to a specific detection moiety in combination with another oligonucleotide probe conjugated to the same detection moiety or a similar detection moiety that generates a detectable signal that can be detected simultaneously with the signal generated by the detectable moiety of the LOCS (e.g., using a single type of detector, such as one fluorescent channel, or a particular mode of colorimetric, surface plasmon resonance (SPR), chemiluminescent, or electrochemical detection).

[0351] Analysis of fluorescent signals By way of example only and not limitation, detectable moieties used in accordance with the present invention include fluorescent signals generated by these detectable moieties after modification, including binding, dissociation of split-LOCS structures, cleavage or digestion of coupled or otherwise associated oligonucleotide probes, which can be analyzed in any suitable manner to detect, differentiate, and / or quantify target molecules in accordance with the methods of the present invention.

[0352] While standard melting curve analysis can be used, a variety of other approaches are disclosed and exemplified herein (see Examples) that can be readily adapted for analysis of a variety of assay formats.

[0353] By way of non-limiting example, measurements of the fluorescent signal at a single temperature or at multiple temperatures can be obtained at various time points in a reaction suitable for detecting cleavage or degradation of the loop region of a LOCS oligonucleotide. By way of non-limiting example, these time points can include (i) at or near the beginning of the reaction, and / or (ii) at a single time point or multiple time points during the course of the reaction, and / or (iii) at the termination or endpoint of the reaction.

[0354] In some embodiments, measurements of the fluorescent signal can be obtained at two or more temperatures at each cycle during an amplification reaction, such as a PCR amplification, and analysis can be performed by comparing the levels of fluorescence obtained at the first and / or second temperatures and / or at further temperatures.

[0355] In some embodiments, measurements of the fluorescent signal can be obtained at two temperatures in reactions tailored to measure two targets at the same wavelength.

[0356] In some embodiments, a first target can be detected using a first oligonucleotide reporter probe or substrate, and the fluorescent signal can be measured at multiple time points or multiple cycles, e.g., each cycle during PCR. A second target can be detected in the same reaction using a LOCS probe by comparing pre- and post-PCR fluorescence levels. In such embodiments, quantitative data can be determined for the first target, while qualitative data can be generated for the second target. As a non-limiting example, a first oligonucleotide probe can be an MNAzyme substrate that is cleaved by a first MNAzyme in the presence of the first target and monitored in real time, while a LOCS probe can be cleaved by a second MNAzyme in the presence of the second target and monitored using an endpoint detection assay. Examples of combining real-time quantitative and endpoint qualitative assays include Example 1, which utilizes one linear MNAzyme substrate and one LOCS probe, and Example 6, which utilizes one TaqMan probe and one LOCS probe.

[0357] In some embodiments, an increase in fluorescence at a first temperature indicates the presence of a first target, and an increase in fluorescence at a second temperature indicates the presence of a first target and / or a second target. In other embodiments, an increase in fluorescence at a first temperature indicates the presence of a first target, and an increase in fluorescence at a second temperature indicates the presence of a second target. In still other embodiments, a decrease in fluorescence at a first temperature indicates the presence of a first target, and an increase in fluorescence at a second temperature indicates the presence of a second target. In other embodiments, measurements of the fluorescent signal can be obtained at two or more temperatures at each cycle during PCR, and an amplification curve can be plotted for each series of measurements obtained at each temperature. A threshold fluorescence value can be assigned to each amplification plot for each particular temperature, and a Cq value can be measured as the cycle number at which the amplification plot crosses the threshold. In embodiments where the first probe is a standard linear MNAzyme substrate for the detection of target 1 and the second probe is a LOCS reporter for the detection of target 2, and measurements of fluorescent signals are obtained at two temperatures at each cycle during PCR, the Cq measured using the fluorescent signal from the linear MNAzyme substrate at a lower temperature that is below the split LOCS Tm can allow direct quantification of the starting concentration of the first target, and the Cq measured using the total fluorescent signal from both the linear MNAzyme substrate and the LOCS reporter at a higher temperature that is above the Tm of the split LOCS but below the Tm of the intact LOCS can be analyzed as exemplified in Example 4, thus allowing quantification of the starting concentration of the second target.

[0358] In other embodiments, measurements of fluorescent signals can be obtained at two or more temperatures at each cycle during PCR, and amplification curves can be plotted for each series of measurements obtained at each temperature. A threshold fluorescent value can be assigned to each amplification plot for each specific temperature, and a Cq value can be measured as the cycle number at which the amplification plot crosses the threshold. In embodiments where a first probe for detecting a first target, which is a standard molecular beacon, a catcher / pitcher probe, a Scorpion uniprobe, or a Scorpion biprobe, is combined with a second probe for detecting a second target, which may be a LOCS reporter, and measurements of fluorescent signals are obtained at two temperatures at each cycle during PCR, the Cq measured using the fluorescent signal from the first probe at a lower temperature below the Tm of the split LOCS can allow for direct quantification of the starting concentration of the first target, and the Cq measured using the fluorescent signal from the LOCS reporter at a higher temperature above the Tm of the split LOCS but below the Tm of the intact LOCS can allow for direct quantification of the starting concentration of the second target. Particular combinations of a first standard probe and a second LOCS probe may have additional requirements for the Tm of a particular region of the first probe and the two temperatures at which data are acquired, as outlined above for the molecular beacons in Examples 5 and 7, the Scorpion probe in Example 9, and the Catcher-Pitcher oligonucleotide in Example 11.

[0359] As a non-limiting example, a baseline fluorescent signal can be obtained by measuring fluorescence at selected temperatures, e.g., a first and second temperature, at or near the beginning of the reaction, e.g., before PCR. At the first temperature, the standard reporter probe, e.g., linear MNAzyme substrate, TaqMan probe or molecular beacon, and intact LOCS are quenched prior to PCR, and if this temperature is below the Tm of the stem of the intact LOCS (and molecular beacon, if present), no significant fluorescent signal is generated. Analysis can be performed by comparing the levels of fluorescence obtained at the first and second temperatures at the beginning of the reaction (e.g., before PCR) with the levels of fluorescence obtained at the first and second temperatures at a time point during and / or after the reaction (e.g., during or after PCR).

[0360] As demonstrated in Example 1, cleavage of a linear MNAzyme measured at a first temperature and post-PCR time point generates a significant fluorescent signal compared to the signal of the intact linear MNAzyme substrate measured at a first temperature and pre-PCR time point. This relative signal (ΔS D1 ) exceeds a first predetermined threshold, indicating the presence of the first target in the sample. At this first temperature, the intact LOCS and / or the cleaved split LOCS do not contribute to the generation of a significant fluorescent signal compared to the signal obtained before PCR, due to the Tm of the stems of both LOCS structures. At a second temperature higher than the first temperature and at a post-PCR time point, cleavage of the LOCS generates a significant fluorescent signal compared to the signal obtained at the second temperature and at the pre-PCR time point. This relative signal (ΔS D2 ) exceeds a second predetermined threshold and the first temperature (ΔS D1), thus indicating detection of the split LOCS and thus the presence of the second target. At this second temperature and post-PCR time point, the intact LOCS do not contribute to the generation of a significant fluorescent signal compared to the pre-PCR time point at the same second temperature, and do not exceed a predetermined threshold. Alternatively, the difference (ΔS) between the relative signal obtained before and after PCR at the second temperature and the relative signal obtained before and after PCR at the first temperature can be calculated. D2 -ΔS D1 ) can be compared to a predetermined threshold to determine the presence of cleaved split LOCS. As demonstrated in Example 1 (Endpoint Analysis Method 2), if the difference is greater than a predetermined threshold (ΔS D2 -ΔS D1 >threshold), the presence of cleaved split LOCS, indicating the presence of a second target in the sample, can be determined. D2 -ΔS D1 <threshold), the absence of the second target in the sample can be determined. Furthermore, the difference (ΔS) between the relative signal obtained before and after PCR at the second temperature and the relative signal obtained before and after PCR at the first temperature can be determined. D2 -ΔS D1 ) exceeds a predetermined threshold, the intrinsic ratio of the fluorescence change at temperature 1 to temperature 2 (ΔS D1 :ΔS D2 ), or the inverse ratio (ΔS D2 :ΔS D1 ) can be used to determine whether target 1, target 2, or both targets 1 and 2 are present in a sample. As demonstrated in Example 1 (Endpoint Assay Method 3), ΔS D1 :ΔS D2 If is greater than the threshold R1, this indicates that only target 1 is present, and ΔS D1 :ΔS D2 If is less than the threshold R2, this indicates that only target 2 is present, and ΔS D1 :ΔS D2 If R is less than threshold R1 but greater than threshold R2, this indicates that both target 1 and target 2 are present.

[0361] Exemplary Uses of LOCS Oligonucleotides in Combination with Other Standard Reporters / Probes - detection of targets during or after target amplification The LOCS oligonucleotides of the present invention can be used to determine the presence of amplified target nucleic acid sequences. There are no specific limitations regarding the amplification technology to which the LOCS reporter can be applied. Amplicons generated by various reactions can be detected by the LOCS reporter, provided that the presence of the target amplicon can prompt cleavage or degradation of the LOCS reporter to generate a split LOCS structure. Non-limiting examples of methods useful for cleaving or degrading the loop region contained within the LOCS structure include exonuclease degradation, including, but not limited to, cleavage by MNAzymes, DNAzymes, ribozymes, restriction enzymes, endonucleases, or the exonuclease activity of polymerases.

[0362] Generally, nucleic acid amplification techniques utilize an enzyme (e.g., a polymerase) to generate copies of a target nucleic acid to which one or more oligonucleotide primers specifically bind. Non-limiting examples of amplification techniques in which LOCS oligonucleotides can be used include polymerase chain reaction (PCR), 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 (TAMP), and transcription-mediated amplification (TAMP). The amplification methods include one or more of: MA, self-sustained sequence replication (3SR), nucleic acid sequence-based amplification (NASBA), ligase chain reaction (LCR) or divergent amplification method (RAM).

[0363] Those skilled in the art will readily appreciate that the above-described uses of LOCS oligonucleotides are provided for non-limiting illustrative purposes only. The disclosed LOCS oligonucleotides can be used in any primer-based nucleic acid amplification technique, and the invention is not so limited to those specifically described embodiments.

[0364] -Detection of amplicons generated using LOCS reporters As discussed above, the LOCS reporters of the present invention can be utilized in any polynucleotide amplification technique, non-limiting examples of which include PCR, RT-PCR, SDA, HDA, RPA, LAMP, RCA, TMA, RAM, LCR, 3SR, or NASBA.

[0365] Amplicons generated by these techniques can be detected utilizing LOCS reporters that can be cleaved or degraded using any suitable method known in the art, non-limiting examples include the use of catalytic nucleic acids, exonucleases, endonucleases, etc.

[0366] MNAzymes can be used to generate split LOCS reporters by detecting amplicons generated through methods such as PCR, RT-PCR, SDA, HDA, RPA, TMA, LAMP, RCA, LCR, RAM, 3SR, and NASBA. MNAzymes can contain one or more partzymes. MNAzymes are multicomponent nucleic acid enzymes that assemble and are catalytically active only in the presence of, for example, an assembly facilitator (which can be a target to be detected, such as an amplicon generated from a polynucleotide sequence using primers). MNAzymes are composed of multiple partial enzymes or partzymes that self-assemble in the presence of one or more assembly facilitators to form an active MNAzyme that catalytically modifies a substrate. The substrate and assembly facilitator (target) are separate nucleic acid molecules. Partzymes have multiple domains, including (i) a sensor arm that binds to the assembly facilitator (such as a target nucleic acid), (ii) a substrate arm that binds to the substrate, and (iii) a partial catalytic core sequence that combines upon assembly to provide a complete catalytic core. MNAzymes can be designed to recognize a wide range of assembly facilitators, including, for example, different target nucleic acid sequences. In response to the presence of the assembly facilitator, MNAzymes modify their substrates. This substrate modification can be associated with signal generation, and thus the MNAzyme can generate an enzymatically amplified output signal. The assembly facilitator can be a target nucleic acid (e.g., an amplicon generated from a polynucleotide target using primers) present in a biological or environmental sample. In such cases, detection of substrate modification by MNAzyme activity indicates the presence of the target. Several MNAzymes capable of cleaving nucleic acid substrates are known in the art. MNAzymes and modified forms thereof are known in the art and are disclosed in PCT Patent Publication Nos. WO / 2007 / 041774, WO / 2008 / 040095, WO2008 / 122084, and related U.S. Patent Publication Nos. 2007-0231810, 2010-0136536, and 2011-0143338 (the entire contents of each of these documents are incorporated herein by reference).

[0367] - Use of the LOCS reporter as an internal calibrator for inter-instrument or inter-well variability The calibrator method demonstrated in Example 12 has several advantages, including that it does not require the use of additional reagents added to the reaction and does not require the use of data obtained from other wells. This method functions to calibrate and correct for well-to-well variations that may exist. Furthermore, the calibration was performed using data acquired in the same channel, but Therefore, it is not affected by any inter-channel variability that may exist between instruments. When multiple channels are utilized for multiplex reactions, each channel can be independently calibrated against its own LOCS calibration signal. This is advantageous in scenarios where signals are calibrated against signals in different channels, such as those from internal or endogenous controls, because calibration would be adversely affected if the ratio of expected signal intensities between channels differed significantly between instruments, causing inter-channel variability.

[0368] -Diagnostic applications Methods using standard reporter probes in combination with LOCS oligonucleotides can be used for diagnostic and / or prognostic purposes, according to the methods described herein. Diagnostic and / or prognostic methods can 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 therefore non-diagnostic or non-prognostic uses are also contemplated.

[0369] In some embodiments, the methods described herein can be used to diagnose an infection in a subject. For example, these methods can be used to diagnose bacterial, viral, fungal / yeast, protozoan, and / or nematode infections in a subject. In one embodiment, the virus can be an enterovirus. The subject can be a bovine, equine, ovine, primate, avian, or rodent species. For example, the subject can be a mammal, such as a human, dog, cat, horse, sheep, goat, or cow. The subject can be suffering from a disease resulting from the infection. For example, the subject can have meningitis resulting from an enterovirus infection. Thus, in certain embodiments, the methods of the present invention can be used to diagnose meningitis.

[0370] The methods of the present invention can be performed on a sample. The sample can be from any source. For example, the sample can be obtained from an environmental source, an industrial source, or by chemical synthesis.

[0371] As contemplated herein, a "sample" is understood to include a sample that has been modified from its original state, for example, by purification, dilution, or the addition of any other component.

[0372] The methods of the present invention, including but not limited to diagnostic and / or prognostic methods, can be performed on a biological sample. The biological sample can be collected from a subject. Archived biological samples can also be used. Non-limiting examples of suitable biological samples include whole blood or its components (e.g., blood cells, plasma, serum), urine, stool, saliva, lymph, bile, sputum, tears, cerebrospinal fluid, bronchoalveolar lavage fluid, synovial fluid, semen, ascites tumor fluid, breast milk, and pus.

[0373] kit The present invention provides kits comprising one or more agents for carrying out the methods of the invention. Typically, kits for carrying out the methods of the invention contain all of the reagents necessary to carry out the method.

[0374] In some embodiments, the kit may include oligonucleotide components capable of forming an MNAzyme in the presence of a suitable assembly facilitator (e.g., an amplicon as described herein). For example, the kit may include at least first and second oligonucleotide components comprising first and second partzymes, and a second container comprising a substrate, wherein self-assembly of the first and second partzymes and substrate into an MNAzyme requires association of an assembly facilitator (e.g., an amplicon) present in a test sample. Thus, in such embodiments, a LOCS oligonucleotide comprising the first and second partzymes and the substrate within the loop region is applied to a test sample to detect the presence of one or more target amplicons. Generally, the kits include at least one LOCS oligonucleotide provided herein.

[0375] Typically, the kits of the invention also include other reagents, washing reagents, enzymes, and / or other reagents required for carrying out the methods of the invention, such as PCR or other nucleic acid amplification techniques.

[0376] The kit may be a fragmented kit or a combined kit as defined herein.

[0377] A fragmented kit includes reagents housed in separate containers, which may include small glass containers, plastic containers, or strips of plastic or paper. Such containers may allow for efficient transfer of reagents from one compartment to another, and the addition of agents or solutions from each container in a quantitative manner from one compartment to another, while avoiding cross-contamination of samples and reagents.

[0378] Such kits may also include a container for receiving the test sample, containers containing reagents used in the assay, containers containing washing reagents, and containers containing detection reagents. A combined kit contains all of the components of a reaction assay in a single container (eg, a single box containing each of the desired components).

[0379] The kits of the invention may also include instructions for using the kit components to perform a suitable method. The kits and methods of the invention can be used in conjunction with automated analytical equipment and systems, including, but not limited to, real-time PCR instruments.

[0380] Applications for the amplification, detection, identification, or quantification of different targets may be applicable to a single kit of the invention, or alternatively, may require, for example, different kits containing reagents specific for each target. The methods and kits of the invention find use in any situation in which it is desirable to detect, identify, or quantitate any entity.

[0381] It will be appreciated by those skilled in the art that numerous variations and / or modifications may be made to the present invention as disclosed in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive. [Example]

[0382] The present invention will now be described in more detail by reference to the following specific examples, which should not be construed in any way as limiting the scope of the invention.

[0383] Example 1 A method for the analysis of multiple targets at a single wavelength using one linear MNAzyme substrate and one LOCS probe in a format that allows either simultaneous real-time quantification of one target combined with qualitative endpoint detection of a second target, or simultaneous qualitative endpoint analysis of two targets per channel. The following example demonstrates an approach in which one linear substrate and one LOCS reporter are used in combination for the detection and differentiation of two targets (CTcry and NGopa) and for the quantification of one target (CTcry) in a single fluorescence channel without the need for melting curve analysis. The assay is designed so that CTcry can be detected and quantified using a linear MNAzyme substrate, and NGopa can be detected and differentiated at the endpoint using a LOCS reporter containing a different MNAzyme substrate within its loop.

[0384] During PCR, MNAzyme 1 can cleave linear substrate 1 in the presence of CTcry, separating the fluorophore and quencher, resulting in an increased signal that can be detected over a wide range of temperatures. In this example, real-time detection and quantification of CTcry is achieved by acquiring fluorescence at each cycle during PCR at Temperature 1 (D1) (52°C). The stem of LOCS-1, both intact and in the split configuration, has a melting temperature (Tm) higher than Temperature 1 (52°C); therefore, LOCS-1 does not contribute to the signal at Temperature 1, regardless of the presence or absence of NGopa in the sample.

[0385] In the presence of NGopa, MNAzyme 2 can cleave LOCS-1 during PCR. End-point detection of NGopa is useful for determining the amplification (ΔS D2 This can be achieved by comparing the fluorescence signal at a higher temperature 2 (70 °C) before and after RT. Because the Tm of the intact LOCS-1 stem is higher than temperature 2 (Tm > 70 °C) and the Tm of the split LOCS-1 stem is lower than temperature 2 (Tm < 70 °C), an increase in fluorescence during PCR above that associated with cleaved linear substrate 1 (if present) at this temperature is associated with split LOCS-1 with the dissociated stem and indicates the presence of NGopa.

[0386] In this example, real-time quantification of target 1 (CTcry) is demonstrated in combination with endpoint detection of target 2 (NGopa). Also in this example, endpoint detection of both target 1 (CTcry) and target 2 (NGopa) is demonstrated, along with several alternative methods of analysis of these data.

[0387] The following endpoint analysis methods (1-3) require measurement of fluorescence only at discrete time points: at or near the beginning of the amplification reaction (pre-PCR fluorescence) and after amplification (endpoint or post-PCR fluorescence). Readings are taken at these time points and at multiple temperatures (D1 and D2), allowing the analysis to reveal the presence of target 1, or target 2, or targets 1 and 2, or the absence of both targets 1 and 2.

[0388] End Point Analysis Method 1 At temperature 1 (D1), target-mediated cleavage of Substrate 1 results in a significant increase in the fluorescent signal during PCR (ΔS D1 ) which results in the post-PCR fluorescence at D1 (S D1-post-PCR ) and pre-PCR fluorescence at D1 (S D1-pre-PCR ) which is measured as the difference between S D1-post-PCR -S D1-pre-PCR =ΔS D1 The first threshold (X1) is exceeded so that >X1. In contrast, at this temperature, if cleavage of LOCS-1 occurs, this does not result in a significant increase in the fluorescent signal, and therefore the threshold (X1) is not exceeded. This occurs because the Tm of the cleaved stem is higher than D1, and the structure remains quenched. Therefore, comparison of pre- and post-PCR measurements of fluorescence at temperature 1 allows for the specific detection of cleaved substrate 1, and thus target 1.

[0389] At temperature 2 (D2), which is higher than temperature 1 (D1), target-mediated cleavage of LOCS-1 leads to a significant increase in the fluorescent signal during amplification. In the presence of split LOCS-1, the signal increase at D2 (ΔS D2 ) is greater than the threshold X1. Furthermore, regardless of whether substrate 1 is cleaved or not, the increase in signal at D2 (ΔS D2) is the signal increase at D1 (ΔS D1 ) is larger than the post-PCR fluorescence at D2 (S D2-post-PCR ) and fluorescence before PCR at D2 (S D2-pre-PCR The increase in fluorescence at D2 (ΔS) is measured as the difference between D2 ) size (S D2-post-PCR -S D2-pre-PCR =ΔS D2 ) is the fluorescence at D1 (ΔS D1 ) and threshold X1 (ΔS D2 >ΔS D1 and ΔS D2 > threshold X1), which indicates the detection of split LOCS-1 and thus the presence of target 2.

[0390] End Point Analysis Method 2 At temperature 1 (D1), cleavage of substrate 1 occurs during PCR with a ΔS D1 A fluorescent signal (ΔS) above a first threshold (X1), such as >X1 D1 ), whereas cleavage of LOCS-1 does not result in a significant increase in the fluorescent signal at D1, which, either intact or cleaved, does not exceed the threshold (X1) due to the high Tm of the stem over D1. Thus, comparison of pre- and post-PCR fluorescence measurements at D1 (ΔSD1) allows for the specific detection of cleaved Substrate 1 and, therefore, Target 1.

[0391] At temperature 2 (D2), which is higher than temperature 1 (D1), the cleavage of LOCS-1 throughout the PCR is significantly smaller than the change (ΔS D1 ) greater than the change in fluorescence signal (ΔS D2 ) and ΔS D2 and ΔS D1 The difference between (ΔS D2 -ΔS D1 ) exceeds the second threshold (X2), and ΔS D2 -ΔS D1 =ΔΔS D2 ΔS D1 >X2. In contrast, cleavage of substrate 1 alone results in a similar ΔS D2 and ΔS D1 values ​​are generated, and the difference between these two values ​​(ΔΔSD2 ΔS D1 ) is not significant and does not exceed the second threshold value (X2). Therefore, the analysis of fluorescence at temperature 2 in this mode enables the specific detection of the cleaved split LOCS-1 indicating the presence of target 2.

[0392] Endpoint analysis method 3 If either target 1 and / or target 2 is present in the sample, the increase in signal (ΔS D2 ) during amplification at temperature 2 is significant and exceeds the threshold value X1. Therefore, when ΔS D2 > X1, this indicates that either CTcry and / or NGopa is present in the sample. Further, when ΔS D2 < X1, this indicates that neither CTcry nor NGopa is present in the sample.

[0393] Furthermore, when ΔS D2 > X1, the specific ratio of fluorescence change from temperature 1 to temperature 2 (ΔS D1 : ΔS D2 ), or the inverse ratio (ΔS D2 : ΔS D1 ) can be used to determine whether target 1, target 2, or both target 1 and 2 are present in the sample.

[0394] ΔS D1 : ΔS D2 being greater than the threshold value R1 indicates that only target 1 is present, ΔS D1 : ΔS D2 being less than the threshold value R2 indicates that only target 2 is present, and ΔS D1 : ΔS D2 being less than the threshold value R1 but greater than the threshold value R2 indicates that both target 1 and target 2 are present.

[0395] Endpoint analysis methods 1, 2 and 3 Furthermore, in the above endpoint analysis methods 1 to 3, the changes in signals (ΔS D1 and ΔS D2) can also be calibrated to the signal from a calibrator (C) or the difference between the post-PCR signal from the calibrator and the pre-PCR signal (ΔC). As a non-limiting example, a calibrator can include an endogenous control, an internal control, or a designated calibrator oligo, which can be measured at a given temperature(s) in the same or different channels. The change in signal at temperatures 1 and 2 (ΔS D1 and ΔS D2 ) as a ratio to the change in signal from the calibrator (e.g., ΔS D2 / ΔC, ΔS D1 / ΔC), or alternatively the inverse ratio (e.g., ΔC / ΔS D2 , ΔC / ΔS D1 ), where ΔC is determined as a positive signal such that ΔC is greater than a threshold C (ΔC > threshold C).

[0396] [Table 4]

[0397] Oligonucleotides Oligonucleotides specific for this experiment include forward primer 1 (SEQ ID NO: 1), reverse primer 1 (SEQ ID NO: 2, partzyme A1 (SEQ ID NO: 3), partzyme B1 (SEQ ID NO: 4), forward primer 2 (SEQ ID NO: 5), reverse primer 2 (SEQ ID NO: 6), partzyme A2 (SEQ ID NO: 7), partzyme B2 (SEQ ID NO: 8), forward primer 3 (SEQ ID NO: 9), reverse primer 3 (SEQ ID NO: 10), partzyme A3 (SEQ ID NO: 11), partzyme B3 (SEQ ID NO: 12), linear MNAzyme substrate 1 (SEQ ID NO: 13), LOCS-1 (SEQ ID NO: 14) and linear MNAzyme substrate 2 (SEQ ID NO: 15). Sequences are listed in the Sequence Listing.

[0398] The oligonucleotides specific for amplification and detection of target 1 (CTcry) are substrate 1, partzyme A1, partzyme B1 (MNAzyme 1), forward primer 1, and reverse primer 1. The oligonucleotides specific for amplification and detection of target 2 (NGopa) are LOCS-1, partzyme A2, partzyme B2 (MNAzyme 2), forward primer 2, and reverse primer 2. The oligonucleotides specific for amplification and detection of the calibrator gene (TFRC) are substrate 2, partzyme A3, partzyme B3 (MNAzyme 3), forward primer 3, and reverse primer 3.

[0399] Reaction conditions Real-time amplification and detection were performed in a total reaction volume of 20 μL using a BioRad® CFX96 thermal cycler. Cycling parameters and fluorescence data acquisition ( The reaction times were 95°C for 2 minutes, 52°C for 15 seconds (DA), 70°C for 15 seconds (DA), 10 cycles of 95°C for 5 seconds and 61°C for 30 seconds (0.5°C decay per cycle), 40 cycles of 95°C for 5 seconds and 52°C for 40 seconds (DA at each cycle), and 1 cycle of 70°C for 15 seconds (DA). All reactions were performed in triplicate and contained 40 nM of each forward primer, 200 nM of each reverse primer, 200 nM of each partzyme A, 200 nM of each partzyme B, 200 nM of each substrate, 200 nM LOCS-1, and 1x SensiFAST buffer (Bioline). Reactions contained either no target (NF HO) or a synthetic G-block of CTcry (20,000, 4,000, 800, 160, or 32 copies), or NGopa gene (20,000 or 32 copies), or various concentrations of CTcry gene (20,000, 4,000, 800, 160, or 32 copies) in a background of NGopa gene (20,000 and 32 copies). All reactions except for HO further contained a background of 34.5 ng (10,000 copies) of human genomic DNA. Finally, an additional control contained only genomic DNA without any CTcry or NGopa G blocks.

[0400] result In this example, three MNAzymes (MNAzymes 1-3) were used in a single PCR to simultaneously detect and differentiate three target nucleic acids (CTcry, NGopa, and human TFRC, respectively) using only two fluorescent channels (HEX and Texas Red). The presence or absence of CTcry and / or NGopa was detected and differentiated in the HEX channel, and the presence of the TFRC gene was detected in the Texas Red channel. The presence of CTcry and TFRC genes was detected in real time by an increase in fluorescent signal at 52°C (D1) in the HEX and Texas Red channels, respectively, and at an endpoint at 52°C. The presence of NGopa was detected using a separate measurement at 70°C (D2) in the HEX channel, monitoring the cleavage of LOCS-1.

[0401] The results shown in Figure 6 show PCR amplification curves obtained in the HEX channel at an acquisition temperature of 52 °C from reactions containing 20,000 (black dots), 4,000 (black dashed line), 800 (black squares), 160 (gray solid line), or 32 (gray dots) copies of CTcry template alone (Figure 6A), or in a background of 20,000 (Figure 6B), or 32 (Figure 6C) copies of NGopa template. Fluorescence data were also collected at 52 °C for reactions lacking CTcry but containing either 20,000 (black line) or 32 (gray line) copies of NGopa template (Figure 6D). A no-target control (NF HO) is shown in Figures 6A–6C (solid black line). Amplification curves are the average of fluorescence levels from triplicate reactions. The calculated copy numbers of CTcry for each of the above reactions are shown in Table 5, with not applicable (N / A) indicating the absence of a Cq value determined at 52°C, consistent with the absence of CTcry in these reactions. Cq values ​​were determined using the regression mode of BioRad software (baseline subtraction curve fit).

[0402] The results in Table 5 show that the CTcry standard curve has an R of greater than 0.99. 2 The PCR efficiency was high (100-106%). The presence of 20,000 or 32 copies of NGopa in the reaction did not significantly change the calculated gene copy number of CTcry (paired Student's t-test, p-values ​​0.144 and 0.315, respectively), thus demonstrating that real-time detection at 52°C can be used for direct quantitative analysis of CTcry in samples.

[0403] [Table 5]

[0404] The results in Fi...

Claims

1. 1. A method for determining the presence or absence of a first and a second target in a sample, comprising: (a) subjecting the sample or a derivative thereof suspected of containing the first and second targets to a first oligonucleotide for the detection of said first target, said first oligonucleotide comprising a first detection moiety capable of generating a first detectable signal, an intact stem-loop oligonucleotide for detection of said second target, comprising a double-stranded stem portion of hybridized nucleotides, the opposing strand of which is linked by an unbroken single-stranded loop portion of unhybridized nucleotides, said stem portion comprising a second detection portion capable of generating a second detectable signal, The first and second detection moieties are capable of producing indistinguishable detectable signals at a single temperature using a single type of detector, and - preparing a mixture for a reaction by contacting with a first enzyme capable of digesting one or more of said unhybridized nucleotides of said intact stem-loop oligonucleotide only if said second target is present in said sample; (b) the mixture; - said first target is suitable for inducing a modification to said first oligonucleotide, thereby enabling said first detection moiety to generate a first detectable signal; - treating under conditions suitable for digesting one or more of said unhybridized nucleotides of said intact stem-loop oligonucleotide with said first enzyme only if said second target is present in said sample, thereby disrupting said single-stranded loop portion to provide a split stem-loop oligonucleotide; (c) - measuring the background signal provided by said first and second detection moieties in said mixture or in a control mixture; (d) at one or more times during or after said processing, - whether a first detectable signal resulting from said modification is produced at the first temperature, said first detectable signal being distinct from said background signal and indicative of the presence of said first target in said sample; - determining whether a second detectable signal is produced at a second temperature that is distinct from the background signal and is indicative of the presence of the second target in the sample. Including, - at said first temperature, said second detectable signal is not different from said background signal; At the second temperature, a strand of the double-stranded stem portion of the split stem-loop oligonucleotide, if present, is partially or completely dissociated, allowing the second detection moiety to provide the second detectable signal; wherein the strands of the double-stranded stem portion of the intact stem-loop oligonucleotide, if present, are unable to dissociate, thereby preventing the second detectable moiety from providing the second detectable signal.

2. The determination in part (d) is - using a pre-defined threshold value to determine whether the first detectable signal resulting from the modification is different from any of the background signals at the first temperature, and / or The method of claim 1, comprising using a predetermined threshold value to determine whether the second detectable signal is different from any of the background signals at the second temperature.

3. - said modification to said first oligonucleotide enables said first detection moiety to provide said first detectable signal at or below said first temperature; - the production of said first detectable signal is reversible; Part (c) measuring a first background signal within 5° C. of a first temperature and a second background signal within 5° C. of a second temperature; These are provided by the first and second detection moieties in the mixture or in the control mixture, - part (d) at one or more times during or after said processing, whether a first detectable signal resulting from the modification is produced at the first temperature, the first detectable signal being distinct from the first background signal and indicative of the presence of the first target in the sample; 2. The method of claim 1, comprising determining whether a second detectable signal is produced at the second temperature that is distinct from the second background signal and that indicates the presence of the second target in the sample.

4. - said first target is a nucleic acid sequence, - said first oligonucleotide is a stem-loop oligonucleotide comprising a double-stranded stem portion of hybridized nucleotides on opposing strands linked by an unbroken single-stranded loop portion of unhybridized nucleotides, all or part of which is complementary to said first target, - said modification of said first oligonucleotide is a conformational change resulting from hybridization of said target to said single-stranded loop portion of said first oligonucleotide by complementary base pairing, - said conformational change is a strand dissociation of the double-stranded stem portion of said first oligonucleotide resulting from said hybridization of said target to said single-stranded loop portion of said first oligonucleotide by complementary base pairing; (a) - the stem portion of the first oligonucleotide is below the melting temperature (Tm) of the double-stranded duplex structure formed from the hybridization of the target to the single-stranded loop portion of the first oligonucleotide, and having a Tm greater than the Tm of the stem portion, - the double-stranded duplex structure has a Tm that is greater than the Tm of the stem portion of the intact stem-loop oligonucleotide and greater than the Tm of the stem portion of the split stem-loop oligonucleotide; - the stem portion of the intact stem-loop oligonucleotide has a Tm that is greater than the Tm of the stem portion of the split stem-loop oligonucleotide; - the first temperature is below the Tm of the double-stranded duplex, the stem portion of the first oligonucleotide, 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 double-stranded duplex, the stem portion of the first oligonucleotide, and the stem portion of the intact stem-loop oligonucleotide, and above the Tm of the stem portion of the split stem-loop oligonucleotide; - said first temperature is less than said second temperature, (b) - the stem portion of the first oligonucleotide has a melting temperature (Tm) that is less than the melting temperature (Tm) of a double-stranded duplex formed from the hybridization of the target to the single-stranded loop portion of the first oligonucleotide and is less than the Tm of the stem portion of the intact stem-loop oligonucleotide; - the double-stranded duplex structure has a Tm that is greater than the Tm of the stem portion of the split stem-loop oligonucleotide; - the stem portion of the intact stem-loop oligonucleotide has a Tm that is greater than the Tm of the stem portion of the split stem-loop oligonucleotide; - the first temperature is below the Tm of the double-stranded duplex, the stem portion of the first oligonucleotide, the stem portion of the intact stem-loop oligonucleotide, and the stem portion of the split stem-loop oligonucleotide; - the second temperature is above the Tm of the stem portion of the first oligonucleotide and the stem portion of the split stem-loop oligonucleotide, and below the Tm of the double-stranded duplex and the stem portion of the intact stem-loop oligonucleotide; - said first temperature is less than said second temperature, (c) - the stem portion of the first oligonucleotide has a melting temperature (Tm) that is less than the melting temperature (Tm) of a double-stranded duplex formed from the hybridization of the target to the single-stranded loop portion of the first oligonucleotide, less than the Tm of the stem portion of the intact stem-loop oligonucleotide, and less than the Tm of the stem portion of the split stem-loop oligonucleotide; - the double-stranded duplex structure has a Tm that is less than the Tm of the stem portion of the intact stem-loop oligonucleotide; - the stem portion of the intact stem-loop oligonucleotide has a Tm that is greater than the Tm of the stem portion of the split stem-loop oligonucleotide; - the first temperature is below the Tm of the double-stranded duplex, the stem portion of the first oligonucleotide, the stem portion of the intact stem-loop oligonucleotide, and the stem portion of the split stem-loop oligonucleotide; - the second temperature is above the Tm of the double-stranded duplex, the stem portion of the first oligonucleotide, and the stem portion of the split stem-loop oligonucleotide, and below the Tm of the stem portion of the intact stem-loop oligonucleotide; - the first temperature is less than the second temperature, or (d) - the stem portion of the first oligonucleotide is below the melting temperature (Tm) of the double-stranded duplex structure formed from the hybridization of the target to the single-stranded loop portion of the first oligonucleotide, has a Tm that is greater than the Tm of a stem portion and greater than the Tm of the stem portion of the split stem-loop oligonucleotide; - the double-stranded duplex structure has a Tm that is greater than the Tm of the stem portion of the intact stem-loop oligonucleotide and greater than the Tm of the stem portion of the split stem-loop oligonucleotide; - the stem portion of the intact stem-loop oligonucleotide has a Tm that is greater than the Tm of the stem portion of the split stem-loop oligonucleotide; - said first temperature is below the Tm of said stem portion and said double-stranded duplex of said first oligonucleotide and above the Tm of said stem portion of said intact stem-loop oligonucleotide and said stem portion of said split stem-loop oligonucleotide; - the second temperature is below the Tm of the stem portion of the first oligonucleotide, the double-stranded duplex, and the stem portion of the intact stem-loop oligonucleotide, and above the Tm of the stem portion of the split stem-loop oligonucleotide; The method of claim 3, wherein the first temperature is greater than the second temperature.

5. - said first target is a nucleic acid sequence, said first oligonucleotide comprises a double-stranded stem portion of hybridized nucleotides, the opposing strand of which is connected by a single-stranded loop portion of unhybridized nucleotides, all or a portion of which is complementary to said first target, and a second single-stranded portion extending in the 3' direction from one of the opposing strands and terminating in a sequence complementary to a portion of said first target; a blocker molecule preceding said sequence complementary to said portion of said first target; - the mixture further comprises a polymerase, - said processing said mixture hybridizing the second single-stranded portion to the first target by complementary base pairing; extending the second single-stranded portion using the polymerase and the first target as a template sequence to provide a double-stranded nucleic acid, wherein the blocker molecule prevents the polymerase from using the stem portion of the first oligonucleotide as a template to extend the first target; denaturing the double-stranded nucleic acid and providing the modification to the first oligonucleotide that enables the second single-stranded portion extended by the polymerase to hybridize to the single-stranded loop portion of the first oligonucleotide by complementary base pairing to generate a signaling duplex structure, thereby enabling the first detection moiety to provide the first detectable signal; (a) - the stem portion of the first oligonucleotide has a melting temperature (Tm) that is less than the Tm of the signaling duplex and greater than the Tm of the stem portion of the split stem-loop oligonucleotide; - the signaling duplex has a Tm that is greater than the Tm of the stem portion of the intact stem-loop oligonucleotide and greater than the Tm of the stem portion of the split stem-loop oligonucleotide; - the stem portion of the intact stem-loop oligonucleotide has a Tm that is greater than the Tm of the stem portion of the split stem-loop oligonucleotide; - the first temperature is below the Tm of the signaling duplex, the stem portion of the first oligonucleotide, the stem portion of the intact stem-loop oligonucleotide, and the stem portion of the split stem-loop oligonucleotide; - the second temperature is applied to the signaling duplex, the first oligonucleotide the stem portion, and less than the Tm of the stem portion of the intact stem-loop oligonucleotide and greater than the Tm of the stem portion of the split stem-loop oligonucleotide; - said first temperature is less than said second temperature, (b) - the stem portion of the first oligonucleotide has a melting temperature (Tm) that is less than the Tm of the signaling duplex and less than the Tm of the stem portion of the intact stem-loop oligonucleotide; - the signaling duplex has a Tm that is greater than the Tm of the stem portion of the split stem-loop oligonucleotide; - the stem portion of the intact stem-loop oligonucleotide has a Tm that is greater than the Tm of the stem portion of the split stem-loop oligonucleotide; - the first temperature is below the Tm of the signaling duplex, the stem portion of the first oligonucleotide, the stem portion of the intact stem-loop oligonucleotide, and the stem portion of the split stem-loop oligonucleotide; - the second temperature is above a Tm of the stem portion of the first oligonucleotide and the stem portion of the split stem-loop oligonucleotide, and below a Tm of the signaling duplex and the stem portion of the intact stem-loop oligonucleotide; - said first temperature is less than said second temperature, (c) - the stem portion of the first oligonucleotide has a melting temperature (Tm) that is less than the Tm of the signaling duplex, less than the Tm of the stem portion of the intact stem-loop oligonucleotide, and less than the Tm of the stem portion of the split stem-loop oligonucleotide; - the signaling duplex has a Tm that is less than the Tm of the stem portion of the intact stem-loop oligonucleotide; - the stem portion of the intact stem-loop oligonucleotide has a Tm that is greater than the Tm of the stem portion of the split stem-loop oligonucleotide; - the first temperature is below the Tm of the signaling duplex, the stem portion of the first oligonucleotide, the stem portion of the intact stem-loop oligonucleotide, and the stem portion of the split stem-loop oligonucleotide; - the second temperature is above the Tm of the signaling duplex, the stem portion of the first oligonucleotide, and the stem portion of the split stem-loop oligonucleotide, and below the Tm of the stem portion of the intact stem-loop oligonucleotide; - the first temperature is less than the second temperature, or (d) - the stem portion of the first oligonucleotide has a melting temperature (Tm) that is less than the Tm of the signaling duplex, greater than the Tm of the stem portion of the intact stem-loop oligonucleotide, and greater than the Tm of the stem portion of the split stem-loop oligonucleotide; - the signaling duplex has a Tm that is greater than the Tm of the stem portion of the intact stem-loop oligonucleotide and greater than the Tm of the stem portion of the split stem-loop oligonucleotide; - the stem portion of the intact stem-loop oligonucleotide has a Tm that is greater than 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 first oligonucleotide and the signaling duplex, and above 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 first oligonucleotide, the signaling duplex, and the stem portion of the intact stem-loop oligonucleotide; and greater than the Tm of the stem portion of the split stem-loop oligonucleotide; The method of claim 3, wherein the first temperature is greater than the second temperature.

6. - said first detection moiety is a fluorophore and said modification increases its distance from a quencher molecule; - said second detection moiety is a fluorophore, The method of any one of claims 1 to 5, wherein a second detectable signal provided by said strand of the double-stranded stem portion of the split stem-loop oligonucleotide dissociating increases the distance of the fluorophore from the quencher molecule.

7. - said first target is a nucleic acid sequence, said first oligonucleotide comprises a first double-stranded portion of hybridized nucleotides, the first strand extending to a single-stranded portion terminating in a complementary sequence capable of hybridizing to a portion of said first target, said first strand including a blocker molecule preceding said complementary sequence; - the mixture further comprises a polymerase, - said processing said mixture hybridizing the complementary sequence of the single stranded portion to a portion of the first target by complementary base pairing; extending the complementary sequence using the polymerase and the first target as a template sequence to provide a second double-stranded portion, wherein the blocker molecule prevents the polymerase from using the first strand of the first double-stranded portion as a template to extend the first target; denaturing the first and second double-stranded portions; providing said modification to said first oligonucleotide that hybridizes, by complementary base pairing, with said complementary sequence extended by said polymerase onto said first strand of said first double-stranded portion to generate a signaling duplex structure, thereby enabling said first detection moiety to provide said first detectable signal; (a) - the first double-stranded portion has a melting temperature (Tm) that is less than the Tm of the signaling duplex and greater than the Tm of the stem portion of the split stem-loop oligonucleotide; - the signaling duplex has a Tm that is greater than the Tm of the stem portion of the intact stem-loop oligonucleotide and greater than the Tm of the stem portion of the split stem-loop oligonucleotide; - the stem portion of the intact stem-loop oligonucleotide has a Tm that is greater than the Tm of the stem portion of the split stem-loop oligonucleotide; - the first temperature is below the Tm of the signaling duplex, the first double-stranded portion, 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 signaling duplex, the first double-stranded portion, and the stem portion of the intact stem-loop oligonucleotide, and above the Tm of the stem portion of the split stem-loop oligonucleotide; - said first temperature is less than said second temperature, (b) - the first double-stranded portion has a melting temperature (Tm) that is less than the Tm of the signaling duplex and less than the Tm of the stem portion of the intact stem-loop oligonucleotide; - the signaling duplex has a Tm that is greater than the Tm of the stem portion of the split stem-loop oligonucleotide; - the stem portion of the intact stem-loop oligonucleotide has a Tm that is greater than the Tm of the stem portion of the split stem-loop oligonucleotide; - the first temperature is below the Tm of the signaling duplex, the first double-stranded portion, the stem portion of the intact stem-loop oligonucleotide, and the stem portion of the split stem-loop oligonucleotide; - the second temperature is above the Tm of the first double-stranded portion and the stem portion of the split stem-loop oligonucleotide, and below the Tm of the signaling duplex and the stem portion of the intact stem-loop oligonucleotide; - said first temperature is less than said second temperature, (c) - the first double-stranded portion has a melting temperature (Tm) that is less than the Tm of the signaling duplex, less than the Tm of the stem portion of the intact stem-loop oligonucleotide, and less than the Tm of the stem portion of the split stem-loop oligonucleotide; - the signaling duplex has a Tm that is less than the Tm of the stem portion of the intact stem-loop oligonucleotide; - the stem portion of the intact stem-loop oligonucleotide has a Tm that is greater than the Tm of the stem portion of the split stem-loop oligonucleotide; - the first temperature is below the Tm of the signaling duplex, the first double-stranded portion, the stem portion of the intact stem-loop oligonucleotide, and the stem portion of the split stem-loop oligonucleotide; - the second temperature is above the Tm of the signaling duplex, the first double-stranded portion, and the stem portion of the split stem-loop oligonucleotide, and below the Tm of the stem portion of the intact stem-loop oligonucleotide; - the first temperature is less than the second temperature, or (d) - the first double-stranded portion has a melting temperature (Tm) that is less than the Tm of the signaling duplex, greater than the Tm of the stem portion of the intact stem-loop oligonucleotide, and greater than the Tm of the stem portion of the split stem-loop oligonucleotide; - the signaling duplex has a Tm that is greater than the Tm of the stem portion of the intact stem-loop oligonucleotide and greater than the Tm of the stem portion of the split stem-loop oligonucleotide; - the stem portion of the intact stem-loop oligonucleotide has a Tm that is greater than the Tm of the stem portion of the split stem-loop oligonucleotide; - the first temperature is below the Tm of the first double-stranded portion and the signaling duplex, and above 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 first double-stranded portion, the signaling duplex, and the stem portion of the intact stem-loop oligonucleotide, and above the Tm of the stem portion of the split stem-loop oligonucleotide; The method of claim 3, wherein the first temperature is greater than the second temperature.

8. - said first target is a nucleic acid sequence, - the mixture a first primer complementary to a first sequence in the first target; a second oligonucleotide comprising a component complementary to a second sequence in the first target that is different from the first sequence, and a tag portion that is not complementary to the first target; further comprising a first polymerase comprising exonuclease activity; - said processing said mixture conditions suitable for hybridizing said first primer and said second oligonucleotide to said first target; extending the first primer using the first polymerase and the target as a template, thereby cleaving the tag portion; hybridizing the cleaved tag portion to the first oligonucleotide by complementary base pairing; extending the tag portion using the first or second polymerase and the first oligonucleotide as a template to generate a double-stranded sequence comprising the first oligonucleotide, thereby providing the modification to the first oligonucleotide and enabling the first detection moiety to provide the first detectable signal; (a) - said double stranded sequence has a Tm that is greater than the Tm of the stem portion of said intact stem-loop oligonucleotide and greater than the Tm of the stem portion of said split stem-loop oligonucleotide; - the stem portion of the intact stem-loop oligonucleotide has a Tm that is greater than the Tm of the stem portion of the split stem-loop oligonucleotide; - the first temperature is below the Tm of the double-stranded sequence, 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 double-stranded sequence and the stem portion of the intact stem-loop oligonucleotide and above the Tm of the stem portion of the split stem-loop oligonucleotide; - said first temperature is less than said second temperature, (b) - said double stranded sequence has a Tm that is greater than the Tm of said stem portion of said split stem-loop oligonucleotide; - the stem portion of the intact stem-loop oligonucleotide has a Tm that is greater than the Tm of the stem portion of the split stem-loop oligonucleotide; - the first temperature is below the Tm of the double-stranded sequence, the stem portion of the intact stem-loop oligonucleotide, and the stem portion of the split stem-loop oligonucleotide; - the second temperature is above the Tm of the stem portion of the split stem-loop oligonucleotide and below the Tm of the double-stranded sequence and the stem portion of the intact stem-loop oligonucleotide; - said first temperature is less than said second temperature, (c) - said double stranded sequence has a Tm that is less than the Tm of said stem portion of said intact stem-loop oligonucleotide; - the stem portion of the intact stem-loop oligonucleotide has a Tm that is greater than the Tm of the stem portion of the split stem-loop oligonucleotide; - the first temperature is below the Tm of the double-stranded sequence, the stem portion of the intact stem-loop oligonucleotide, and the stem portion of the split stem-loop oligonucleotide; - the second temperature is above the Tm of the double-stranded sequence and the stem portion of the split stem-loop oligonucleotide and below the Tm of the stem portion of the intact stem-loop oligonucleotide; - the first temperature is less than the second temperature, or (d) - said double stranded sequence has a Tm that is greater than the Tm of the stem portion of said intact stem-loop oligonucleotide and greater than the Tm of the stem portion of said split stem-loop oligonucleotide; - the stem portion of the intact stem-loop oligonucleotide has a Tm that is greater than the Tm of the stem portion of the split stem-loop oligonucleotide; - the first temperature is below the Tm of the double-stranded sequence and the intact stem-loop oligonucleotide; above the Tm of the stem portion of the split stem-loop oligonucleotide and the stem portion of the split stem-loop oligonucleotide; - the second temperature is below the Tm of the double-stranded sequence and the stem portion of the intact stem-loop oligonucleotide and above the Tm of the stem portion of the split stem-loop oligonucleotide; The method of claim 3, wherein the first temperature is greater than the second temperature.

9. - said first target is a nucleic acid sequence, - said first oligonucleotide is complementary to a first portion of said target, - the mixture further comprises an additional oligonucleotide complementary to a second portion of the first target, the first and second portions of the first target being adjacent to but not overlapping with each other; - said processing said mixture (i) a first double-stranded moiety by hybridizing said first oligonucleotide to said target by complementary base pairing; and (ii) a second double-stranded component by hybridizing said further oligonucleotide to said target by complementary base pairing; Forming a double structure including providing said modification to said first oligonucleotide, thereby bringing said first and further oligonucleotides into close proximity and enabling said first detection moiety to provide said first detectable signal; - the duplex has a Tm that is less than the Tm of the stem portion of the intact stem-loop oligonucleotide; - the stem portion of the intact stem-loop oligonucleotide has a Tm that is greater than the Tm of the stem portion of the split stem-loop oligonucleotide; - the first temperature is below the Tm of the duplex, the stem portion of the intact stem-loop oligonucleotide, and the stem portion of the split stem-loop oligonucleotide; - the second temperature is above the Tm of the duplex and the stem portion of the split stem-loop oligonucleotide and below the Tm of the stem portion of the intact stem-loop oligonucleotide; - said first temperature is less than said second temperature, - said first detectable moiety is a fluorophore and said further oligonucleotide comprises a quencher, - said forming said duplex structure further brings said fluorophore and quencher into close proximity; The method of claim 3, wherein the detectable signal is a decrease in fluorescence provided by the first detection moiety.

10. (a) - said first target is a nucleic acid sequence, - the first detection moiety is a nanoparticle, a metal nanoparticle, a noble metal nanoparticle, an alkali metal nanoparticle, a gold nanoparticle, or a silver nanoparticle to which the first oligonucleotide is bound; - said processing said mixture introducing said modification to said first oligonucleotide that enables said first detection moiety to hybridize said first target to said first oligonucleotide and provide a first detectable signal indicative of the presence of said first target in said sample; the first detectable signal arises from the first detection moiety following the modification of the first oligonucleotide; (i) a change in the refractive index; (ii) a change in color; and / or (iii) Change in absorption spectrum or (b) - said first target is a nucleic acid sequence, - said first detection moiety is an electrochemical agent to which said first oligonucleotide is attached; - said processing said mixture inducing or promoting said modification to said first oligonucleotide that enables said first detection moiety to hybridize said first target to said first oligonucleotide and thereby provide a first detectable signal indicative of the presence of said first target in said sample; The method of any one of claims 1 to 3, wherein the first detectable signal is a change in an electrochemical signal resulting from the first detection moiety after the modification of the first oligonucleotide.

11. the generation of said first detectable signal is not reversible, - said modification to said first oligonucleotide enables said first detection moiety to provide said first detectable signal at or below said first temperature; - said first detectable signal provided at or below said first temperature remains detectable at said second temperature; Part (c) (i) a first background signal within 1° C., 2° C., 3° ​​C., 4° C., or 5° C. of a first temperature, and a second background signal within 1° C., 2° C., 3° ​​C., 4° C., or 5° C. of a second temperature; and / or (ii) a third background signal at a third temperature which are provided by first and second detection moieties in the mixture or in a control mixture; - part (d) at one or more times during or after said processing, (i) determining whether a first detectable signal resulting from the modification is produced at the first temperature, the first detectable signal being distinct from the first or third background signals; at the first temperature, the second detectable signal is not different from the first or third background signals; determining that detection of a difference between the first detectable signal and the first or third background signal indicates the modification of the first oligonucleotide and the presence of the first target in the sample; and (ii) determining whether a second detectable signal is produced at the second temperature that is distinct from the second or third background signal and that indicates the presence of the second target in the sample.

12. (a) - said first signal is generated which is different from said first background signal; - said second signal is generated which is different from said second background signal; - said second detectable signal differs from said second background signal to a greater extent than said first detectable signal differs from said first background signal; thereby indicating that said second target is present in said sample; or (b) - said first signal is generated which is distinct from said third background signal; - said second signal is generated which is different from said third background signal; - said second signal differs from said third background signal to a greater extent than said first signal differs from said third background signal; thereby indicating that said second target is present in said sample, or (c) - said second temperature is higher than said first temperature, - the third temperature is lower than the Tm of the double-stranded stem portion of the intact stem-loop oligonucleotide; - said first detectable signal is generated which is distinct from said third background signal; - said second detectable signal is generated which is distinct from said third background signal; - said second detectable signal differs from said third background signal to a greater extent than said first signal differs from said third background signal; The method of claim 11 , thereby indicating the presence of the second target in the sample.

13. (a) - the Tm of the stem portion of the intact stem-loop oligonucleotide is greater than the Tm of the stem portion of the split stem-loop oligonucleotide; - said first temperature is less than said second temperature and less than the Tm of said stem portion of said split stem-loop oligonucleotide; - the second temperature is above the Tm of the stem portion of the split stem-loop oligonucleotide and below the Tm of the stem portion of the intact stem-loop oligonucleotide; or (b) - the Tm of the stem portion of the intact stem-loop oligonucleotide is greater than the Tm of the stem portion of the split stem-loop oligonucleotide; - said first temperature is greater than said second temperature, greater than the Tm of said stem portion of said split stem-loop oligonucleotide, and greater than the Tm of said stem portion of said intact stem-loop oligonucleotide; The method of claim 11, wherein the second temperature is above the Tm of the stem portion of the split stem-loop oligonucleotide and below the Tm of the stem portion of the intact stem-loop oligonucleotide.

14. (a) - said first oligonucleotide is a substrate for a multicomponent nucleic acid enzyme (MNAzyme); - the mixture an MNAzyme capable of cleaving said first oligonucleotide if said first target is present in said sample; - said processing said mixture or further comprising binding said MNAzyme to said first target and hybridizing a substrate arm of said MNAzyme to said first oligonucleotide by complementary base pairing that facilitates cleavage of said first oligonucleotide, thereby providing said modification to said first oligonucleotide and enabling said first detection moiety to provide said first detectable signal; (b) - said first oligonucleotide is a substrate for an aptazyme; - said first target is an analyte, a protein, a compound or a molecule; - the mixture further comprises an aptazyme comprising an aptamer capable of binding to the first target, - said processing said mixture or further comprising binding the aptazyme to the first target and the first oligonucleotide to facilitate cleavage of the first oligonucleotide, thereby providing the modification to the first oligonucleotide and enabling the first detection moiety to generate the first detectable signal; (c) - said first target is a nucleic acid sequence, - said first oligonucleotide comprises a sequence that is complementary to said first target, - the mixture a primer complementary to a portion of the first target; and further comprising a polymerase having exonuclease activity, - said processing said mixture hybridizing said primer to said first target by complementary base pairing; hybridizing said first oligonucleotide to said first target by complementary base pairing; providing said modification to said first oligonucleotide that enables said polymerase and said first target to be used as a template sequence to extend said primer, thereby digesting said first oligonucleotide, and said first detection moiety to generate said first detectable signal; or (d) - said first target is a nucleic acid sequence, - the mixture a restriction endonuclease capable of digesting a double-stranded duplex containing said first target; - said processing said mixture hybridizing said first oligonucleotide to said first target by complementary base pairing, thereby forming a double-stranded duplex structure; using said restriction endonuclease to digest said duplex, thereby providing said modification to said first oligonucleotide and enabling said first detection moiety to provide said first detectable signal; or (e) - the mixture further comprises a DNAzyme or ribozyme that requires a cofactor for catalytic activity, - said treatment of said mixture Binding the cofactor to the DNAzyme or ribozyme to catalytically activate it; hybridizing said DNAzyme or ribozyme to said first oligonucleotide by complementary base pairing; and providing said modification to said first oligonucleotide that catalytically activates said DNAzyme or ribozyme, thereby digesting said first oligonucleotide, thereby enabling said first detection moiety to provide said first detectable signal. using conditions suitable for The method of any one of claims 11 to 13, wherein the first target is the cofactor.

15. - said first enzyme is a first MNAzyme; - said processing said mixture The first MNAzyme is allowed to bind to the second target and hybridizing a substrate arm to said loop portion of said intact stem-loop oligonucleotide, thereby digesting said one or more unhybridized nucleotides of said intact stem-loop oligonucleotide to provide said split stem-loop oligonucleotide; - said second target is a nucleic acid sequence, - said processing said mixture The method of any one of claims 1 to 14, further comprising hybridising said second target to said sensor arms of said first MNAzyme by complementary base pairing, thereby promoting assembly of said first MNAzyme.

16. (a) - said second target is a nucleic acid sequence, - said first enzyme is a first restriction endonuclease and said treating said mixture comprises: using conditions suitable for hybridizing said second target to said single-stranded loop portion of said intact stem-loop oligonucleotide by complementary base pairing to form a double-stranded sequence for said first restriction endonuclease to associate with and digest said one or more unhybridized nucleotides of said single-stranded loop portion, thereby forming said split stem-loop oligonucleotide; (b) - the first enzyme comprises a polymerase with exonuclease activity, - said processing said mixture hybridizing said second target to said single-stranded loop portion of said intact stem-loop oligonucleotide by complementary base pairing to form a first double-stranded sequence comprising a portion of said second target; hybridizing a first primer oligonucleotide to the second target to form a second double-stranded sequence located upstream from the first double-stranded sequence that includes the portion of the second target; using the polymerase having exonuclease activity and suitable conditions to extend the primer using the second target as a template sequence; the first polymerase comprising exonuclease activity digests the single-stranded loop portion of the first double-stranded sequence, thereby forming the split stem-loop oligonucleotide; or (c) - said first enzyme is an exonuclease, - said processing said mixture hybridizing said second target to said single-stranded loop portion of said intact stem-loop oligonucleotide by complementary base pairing to form a first double-stranded sequence comprising a portion of said second target; allowing said first enzyme comprising an exonuclease activity to associate with said double-stranded sequence comprising said second target; using conditions suitable for catalytically activating said first enzyme comprising an exonuclease activity to digest said single-stranded loop portion of said first double-stranded sequence comprising said second target, thereby forming said split stem-loop oligonucleotide; or (d) - the first enzyme is a DNAzyme or a ribozyme that requires a cofactor for catalytic activity, and said treating the mixture comprises - binding said cofactor to said first enzyme to catalytically activate it; - binding said DNAzyme or ribozyme to said intact stem-loop oligonucleotide by complementary base pairing; hybridizing to said single-stranded loop portion of the oligonucleotide; - using conditions suitable for catalytically activating said DNAzyme or ribozyme to digest one or more unhybridized nucleotides of said single-stranded loop portion of said intact stem-loop oligonucleotide, thereby forming said split stem-loop oligonucleotide; The method of any one of claims 1 to 14, wherein the second target is the cofactor.

17. The method of any one of claims 1 to 14, wherein said determining the presence or absence of said first and second targets comprises a melting curve analysis.

18. - generating a first target positive control signal using a known concentration of said first target and a known concentration of said modified first oligonucleotide; - generating a first target positive control signal by repeating the method on a separate control sample containing the first target; - generating a second target positive control signal using a known concentration of said second target and a known concentration of said modified stem-loop oligonucleotide; - generating a second target positive control signal by repeating the method on a separate control sample containing the second target, and / or - generating a combined positive control signal by repeating the method on separate control samples containing the first target and the second target; Further comprising: The method according to any one of claims 1 to 14, wherein the combined control sample comprises a known concentration of the first target and / or a known concentration of the second target.

19. (a) (i) the first target, or (ii) the second target; or (iii) assessing the level of a negative control signal by repeating the method of any one of claims 1 to 17 on a separate negative control sample not containing the first target or the second target; - normalizing said first detectable signal and / or said second detectable signal using said negative control signal, and / or (b) further comprising comparing the first and / or second detectable signals to a threshold; - said threshold value is generated using detectable signals derived from a series of samples or derivatives thereof tested according to the method of any one of claims 1 to 17, (i) a no-template control and the first target (ii) a no-template control and the second target (iii) a no-template control, the first target, and the second target. thereby determining the presence or absence of said first and second targets in said sample; The method of claim 18, wherein the series of samples or derivatives thereof are tested using a known concentration of the first oligonucleotide and / or a known concentration of the intact stem-loop oligonucleotide.

20. A method according to any one of claims 1 to 14, wherein said first and second detectable moieties emit in the same colour region of the visible spectrum.

21. a first oligonucleotide for detecting a first target, said first target being a nucleic acid, said first oligonucleotide being complementary to at least a portion of said first oligonucleotide; and a first detection moiety, the first detection moiety is capable of generating a first detectable signal upon modification of the first oligonucleotide; a first detection moiety, wherein the modification is induced by hybridization of the first target to the first oligonucleotide by complementary base pairing; - an intact stem-loop oligonucleotide for the detection of a second target, comprising a double-stranded stem portion of hybridized nucleotides, the opposite strand of which is linked by an unbroken single-stranded loop portion of unhybridized nucleotides, at least one strand of said double-stranded stem portion comprising a second detection moiety; a first enzyme capable of digesting one or more of the unhybridized nucleotides of the intact stem-loop oligonucleotide only if the second target is present in the sample, thereby disrupting the single-stranded loop portion to provide a split stem-loop oligonucleotide; A composition comprising: - said second detection moiety is capable of generating a second detectable signal upon dissociation of said double-stranded stem portion of said split stem-loop oligonucleotide; A composition, wherein said first and second detection moieties are capable of producing indistinguishable detectable signals at a single temperature using a single type of detector.

Images