Multiplex detection of nucleic acids

Abstract

The present invention provides oligonucleotides and methods for detecting and / or distinguishing target nucleic acids. The oligonucleotides and methods are found to be particularly suitable for simultaneously amplifying, detecting and / or distinguishing 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 detecting and / or distinguishing target nucleic acids. The oligonucleotides and methods have been found to be particularly suitable for simultaneously amplifying, detecting, and / or distinguishing multiple targets.

[0002] Incorporated by cross-reference

[0003] This application claims priority to Australian provisional application No. 2018902915 filed on 9 August 2018, the entire contents of which are incorporated herein by cross-reference. Background Art

[0004] Genetic analysis is becoming a routine procedure in the clinic for assessing disease risk, diagnosing disease, predicting patient prognosis or treatment response, and monitoring patient progress. The introduction of such genetic tests relies on the development of simple, inexpensive, and rapid assays for identifying genetic variants.

[0005] In vitro nucleic acid amplification methods have a wide range of applications in genetics and disease diagnosis. Such methods include polymerase chain reaction (PCR), strand displacement amplification (SDA), helicase-dependent amplification (HDA), recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), rolling circle amplification (RCA), transcription-mediated amplification (TMA), self-sustaining sequence replication (3SR), nucleic acid sequence-based amplification (NASBA) or reverse transcription polymerase chain reaction (RT-PCR). Each of these target amplification strategies requires the use of oligonucleotide primers. The amplification process results in an exponential amplification of the amplicon, which incorporates the oligonucleotide primer at its 5' end and contains a newly synthesized copy of the sequence located between the primers.

[0006] Common methods for monitoring the accumulation of amplicons in real time or at the end of amplification include detection using: MNAzymes with universal substrate probes; target-specific molecular beacons, TaqMan probes, or hydrolysis probes; scorpion primers / 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 several of these protocols to obtain additional information. Such protocols measure melting curves resulting from 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 from a complementary target-specific probe labeled with a fluorophore and a quencher. Melting curve analysis provides information about the dissociation kinetics of the two DNA strands during heating. The melting temperature (Tm) is the temperature at which 50% of the DNA dissociates. The Tm depends on the length of the paired nucleotides, the sequence composition, and the GC content. Elucidating information about the target DNA from melting curve analysis typically involves a series of fluorescence measurements, typically taken at small intervals over a wide temperature range. The melting temperature does not depend solely on the base sequence. The melting temperature can be affected by the concentration of the oligonucleotide, the cations in the buffer (monovalent (Na+) and divalent (Mg2+) salts), and the presence or absence of destabilizing agents such as urea or formamide.

[0007] The number of available fluorescence channels capable of monitoring discrete wavelengths is generally limited to the number of targets that can be detected and clearly identified in a single reaction on a real-time instrument. Recently, a scheme known as "labeled oligonucleotide cleavage and extension" (TOCE) has expanded this capability, allowing the analysis of multiple targets at a single wavelength. The TOCE technology uses a pitcher oligonucleotide and a catcher oligonucleotide. The pitcher has two regions - a targeting portion that is complementary to the target and a non-complementary labeling portion located at the 5' end. The capture oligonucleotide is dual-labeled and has a region at its 3' end that is complementary to the labeling portion of the pitcher. During amplification, the pitcher binds to the amplicon, and when the primer is extended, the exonuclease activity of the polymerase can cleave the labeling portion from the pitcher. The released labeling portion then binds to the catcher oligonucleotide and acts as a primer for synthesizing a complementary chain. The melting temperature (catcher Tm) of the double-stranded catcher molecule then serves as a surrogate marker for the original template. Because multiple catchers of different sequences and lengths can be incorporated, all melting at different temperatures, a range of catcher Tm values ​​indicative of a range of targets can be obtained while still measuring at a single wavelength. Limitations of this approach include inherent complexity, as it requires the released fragments to initiate and complete a second extension of the artificial target.

[0008] Hairpin probes or stem-loop probes have also been shown to be useful tools for detecting nucleic acids and / or monitoring target amplification. Hairpin probes dual-labeled with a fluorophore and a quencher dye pair are commonly referred to in the art as molecular beacons. These molecules typically 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, due to complementarity, the loop region binds to the amplicon, and this causes the stem to open, thereby separating the fluorophore-quencher dye pair. The separation of the dye pair attached to the end of the intact, open molecular beacon causes a change in fluorescence that indicates the presence of the target. The method is often used to perform multiplex analysis of multiple targets in a single PCR test. In a multiplex reaction, each molecular beacon has a different target-specific loop region and a unique fluorophore, allowing the hybridization of each different molecular beacon to each amplicon species to be monitored in a separate channel, i.e., at a separate wavelength.

[0009] The concept of molecular beacon has been expanded in the strategy referred to as sloppy beacon (Sloppy Beacon). In this scheme, the loop region of a single beacon is long enough so that it can tolerate mispaired bases and therefore bind to many closely related targets that differ by one or more nucleotides. After amplification, based on the temperature of the loop region separation (melting) of different target species and beacon, melting curve analysis is performed and target species can be distinguished. In this way, multiple closely related species can be detected under a single wavelength and distinguished simultaneously by characterizing the melting curve graph of a specific target with a single sloppy beacon. The difference between standard molecular beacons and sloppy beacons and TaqMan probes and hydrolysis probes is that they are not intended to degrade or crack during amplification. The shortcoming of the technology based on DNA hybridization such as sloppy beacons and TOCE is that it may produce false positive results due to the non-specific hybridization between probe and non-target nucleic acid sequence.

[0010] Many nucleic acid detection assays utilize melting curve analysis to identify the presence of a specific target sequence in a given sample. The melting curve analysis protocol requires measuring fluorescence at various temperatures within an incrementally increasing temperature range. The slope of this curve is then plotted against the temperature to obtain the melting curve. This process is often slow and typically takes anywhere between 30-60 minutes to complete. In addition, melting curve analysis may require skilled personnel to interpret and / or use specialized software for result interpretation. Therefore, there is an urgent need for faster and / or simpler alternatives to melting curve analysis.

[0011] There is a need for improved compositions and methods for the simultaneous detection, differentiation, and / or quantification of multiple unrelated amplicons generated by PCR or by alternative target amplification schemes. Summary of the Invention

[0012] The present invention addresses one or more deficiencies present in current multiplex detection assays.

[0013] Provided herein are methods and compositions for expanding multiplex capabilities during amplification protocols by using oligonucleotide structures referred to herein as LOCS (loops connected to stems). After the LOCS are opened by cracking or degradation in response to the presence of a target, a series of LOCS labeled with a single fluorophore and quencher can be distinguished by the temperature at which the stem portion melts. Therefore, the melting temperature of the stem region serves as a surrogate marker for the specific target of the LOCS. Although other methods in conjunction with stem-loop structures have utilized the following fluorescence changes: a) hybridization of the ring region with the target amplicon (molecular beacon and sloppy beacon), which increases the distance between the dye pair, or b) physical separation of the dye (cleavable molecular beacon) is allowed by cracking, the present invention provides improvements to existing multiplex detection assays. These improvements are at least partially produced by manipulating the melting temperature of the stem portion of the stem-loop oligonucleotide by changing the length and / or sequence composition of the stem so that each stem melts at different temperatures.

[0014] As described herein, multiple LOCS labeled with the same fluorophore can contain: a) different loop sequences, which allow for simultaneous direct or indirect detection of multiple targets, and b) different stem sequences, which melt at discrete temperatures and can be used to identify one or more specific targets present within the multiple targets under investigation. The methods of the present invention have one or more advantages over methods known in the art (e.g., TOCE protocols) because no separate catcher molecule is required, thereby reducing the number of components in the reaction mixture and reducing costs. In addition, the TOCE method is inherently more complex than the method of the present invention because it requires released fragments to initiate and complete the second extension on the synthetic target. In addition, in some embodiments, the LOCS probe can be universal (independent of the target sequence) and / or can be combined with a variety of detection techniques, thereby providing broad applicability in the field of molecular diagnostics. In addition, the melting temperatures used in conventional amplification and detection techniques are based on hybridization and melting of the probe with the target nucleic acid. This has the disadvantage of increased false positives due to non-specific hybridization between the probe and non-target nucleic acid sequences. The methods of the present invention overcome this limitation because the LOCS reporter probe containing the universal substrate does not bind to the target sequence. Finally, it is well known in the art that intramolecular bonds are stronger than intermolecular bonds, and therefore, the likelihood of these uncleaved (closed) LOCS hybridizing to non-specific targets and generating false positive signals is significantly reduced.

[0015] Since intramolecular bonds are stronger than intermolecular bonds, dual-labeled LOCS will melt at a certain temperature when intact (closed), but will melt at a lower temperature after the loop region is opened by target-dependent cleavage or degradation. This property of nucleic acids is exploited in the present invention to extend the ability of the instrument to distinguish multiple targets using a single type of detector (e.g., one fluorescent channel).

[0016] 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 the measurement results of the fluorescent signal generated at a selected temperature rather than a complete temperature gradient, thereby providing advantages in reducing the running time of thermal cycling devices (such as PCR devices). By way of non-limiting example, on a Bio-Rad CFX96 PCR system, a traditional melting analysis with 0.5°C increments and a 5 second holding time at a temperature set between 20°C and 90°C requires 141 cycles of fluorescence measurement and a running time of approximately 50 minutes. Using the LOCS probe, information about the target DNA can be obtained from the same device with 2-6 fluorescence measurements and a running time of approximately 2-5 minutes. Without any specific limitations, reducing the running time may be advantageous in many applications (including, for example, diagnosis).

[0017] The LOCS probes of the present invention can also be used to simultaneously detect, distinguish and / or quantify multiple targets in a single fluorescent channel. In conventional qPCR, the quantification of target DNA is determined using a cycle quantification (Cq) value, which is derived from an amplification curve obtained by measuring fluorescence at a single temperature in each amplification cycle. The Cq value is proportional to the negative logarithm of the concentration of the target DNA, and therefore the concentration can be determined based on the experimentally determined Cq value. However, when there is more than one target-specific probe in a single channel, it is difficult to accurately and specifically quantify each target because it is difficult to identify whether the signal originates from a specific probe. To solve this problem, LOCS is able to accurately and specifically quantify more than one target in a single channel, provided that the amplification curve is obtained by measuring fluorescence at more than one temperature during amplification. This is possible because different LOCS can produce significantly different amounts of fluorescence at different temperatures.

[0018] In embodiments where analysis requires fluorescence acquisition only at a limited number of time points within PCR (e.g., post-PCR), use of the LOCS architecture eliminates the need for acquisition in every cycle. Thus, these embodiments are well suited for very fast cycling protocols that can reduce production time.

[0019] As described above, the melting curve analysis protocol requires measuring fluorescence at various temperatures over a gradually increasing temperature range (e.g., between 30°C and 90°C). The slope of this curve is then plotted as a function of temperature to obtain the melting curve. This process is typically slow and may take, for example, 30-60 minutes to complete. Increasing the speed of melting curve analysis requires the use of highly specialized instruments and cannot be accomplished using standard PCR equipment. Therefore, there is an urgent need for a faster alternative to melting curve analysis that can use standard instruments to simultaneously detect multiple targets in a single fluorescent channel. The melting temperature (Tm) of the LOCS structure of the present invention is predetermined and constant (i.e., not affected by target sequence or concentration), and therefore does not need to be gradually increased over the entire temperature gradient. Each LOCS structure only requires one fluorescence measurement at its specific Tm, without the need to run a complete temperature gradient, thereby facilitating faster results and thus overcoming the above-mentioned limitations.

[0020] Furthermore, melting curve analysis usually requires expert technical personnel or specialized software for result interpretation.

[0021] In some embodiments of the present invention, the use of a single temperature fluorescence measurement after completion of PCR eliminates the need for subjective interpretation of melt curves and facilitates objective determination of the presence or absence of a target.

[0022] In other embodiments of the invention, analysis may require fluorescence acquisition at only a limited number of points within PCR (e.g., post-PCR), eliminating the need for acquisition in every cycle. Thus, these embodiments are well suited for very fast cycling protocols that can reduce production time.

[0023] Several methods have been described that involve fluorescence acquisition at multiple temperatures during PCR (including two temperature acquisitions) to help distinguish between perfectly matched and mismatched probes. In addition, when two targets are present and detection is performed from a single channel, some protocols use multiple acquisition temperatures after each PCR cycle to quantify the concentration of each target. Other methods for simultaneous quantification of two targets can be achieved by performing a complete melt curve at the end of each PCR cycle.

[0024] The LOCS structure of the present invention is compatible with most and potentially all of these existing analytical methods.

[0025] The present invention relates, at least in part, to the following Examples 1-59:

[0026] Example 1. A method for determining the presence or absence of a first target and a second target in a sample, the method comprising:

[0027] - preparing a reaction mixture by contacting a sample, or a derivative thereof, presumably comprising said first target and / or said second target or an amplicon thereof, with:

[0028] a first closed stem-loop oligonucleotide and a second closed stem-loop oligonucleotide, wherein each of the closed stem-loop oligonucleotides comprises a double-stranded stem portion of hybridized nucleotides joined to a closed single-stranded loop portion of unhybridized nucleotides, wherein:

[0029] The first closed stem-loop oligonucleotide and the second closed stem-loop oligonucleotide differ in the number of hybridizing nucleotides and / or the sequence of hybridizing nucleotides in the stem portion, and

[0030] Each of the double-stranded stem portions comprises a fluorophore molecule attached to one strand and a quencher molecule attached to the opposite strand; and

[0031] an enzyme capable of cleaving or degrading the single-stranded loop portions of the first and second closed stem-loop oligonucleotides only when in contact with the target or an amplicon thereof;

[0032] - Working up the reaction mixture:

[0033] under conditions suitable for said enzyme to induce cleavage or degradation of said loop portions of said first closed stem-loop oligonucleotide and said second closed stem-loop oligonucleotide to thereby produce first and second open stem-loop oligonucleotides;

[0034] - detecting the presence or absence of the first open oligonucleotide and the second open oligonucleotide by treating the reaction mixture or a derivative thereof under conditions such that: at a first temperature, strand dissociation of the double-stranded stem portion of the first open-loop stem oligonucleotide occurs at the first temperature, thereby promoting spatial separation of the fluorophore molecule and the quencher molecule of the stem portion of the first open stem-loop oligonucleotide and providing a first detectable signal, and

[0035] at a second temperature, strand dissociation of the double-stranded stem portion of the second open-loop stem oligonucleotide at the second temperature, thereby promoting spatial separation of the fluorophore molecule and the quencher molecule of the stem portion of the second open stem-loop oligonucleotide and providing a second detectable signal,

[0036] in:

[0037] the first temperature is different from the second temperature,

[0038] The fluorophore of the first open stem-loop oligonucleotide and the fluorophore of the second open stem-loop oligonucleotide emit in the same color region of the visible spectrum, and

[0039] Detection of a signal at the first temperature indicates the presence of the first target in the sample, and absence of a signal detected at the first temperature indicates the absence of the first target in the sample; and

[0040] Detection of a signal at the second temperature indicates the presence of the second target in the sample, and absence of a signal detected at the second temperature indicates the absence of the second target in the sample.

[0041] Example 2. The method of embodiment 1, wherein the enzyme comprises a multicomponent nucleic acid enzyme (MNAzyme), and the treating of the reaction mixture comprises treating the reaction mixture under conditions suitable for:

[0042] A first multicomponent nucleic acid enzyme (MNAzyme) binds to the first target or an amplicon thereof and the substrate arm of the first MNAzyme hybridizes to the loop portion of the first closed stem-loop oligonucleotide to thereby promote said cleavage of the loop portion of the first closed stem-loop oligonucleotide by the first MNAzyme to form the first open stem-loop oligonucleotide.

[0043] Example 3 . The method of embodiment 2, wherein the target is a nucleic acid sequence or an amplicon thereof, which is capable of hybridizing to the sensor arm of the first MNAzyme, thereby promoting assembly of the first MNAzyme.

[0044] Example 4. The method of embodiment 1, wherein:

[0045] - the target is an analyte, protein, compound or molecule;

[0046] - the enzyme comprises an enzyme having an aptamer, the aptamer being capable of binding to the first target; and

[0047] - The binding of the first target to the aptamer enables the enzyme having the aptamer to exhibit catalytic activity.

[0048] Example 5 .The method according to embodiment 4, wherein the enzyme having an aptamer comprises any one or more of the following: apta-DNAzyme, apta-ribozyme, apta-MNAzyme.

[0049] Example 6 The method of any one of embodiments 2, 4 or 5, wherein:

[0050] - the target is an analyte, protein, compound or molecule;

[0051] - the reaction mixture further comprises an oligonucleotide sequence capable of hybridizing to the sensor arm of the first MNAzyme to thereby facilitate assembly of the first MNAzyme;

[0052] - said first MNAzyme comprises an aptamer sequence capable of binding to said first target; and

[0053] - Binding of the target to the aptamer enables the first MNAzyme to exhibit catalytic activity.

[0054] Example 7 The method of any one of embodiments 1 to 6, wherein the enzyme comprises a restriction endonuclease, and the treating of the reaction mixture comprises:

[0055] The reaction mixture is treated under conditions suitable for hybridization of the first target or an amplicon thereof to the loop portion of the first closed stem-loop oligonucleotide to form a double-stranded sequence for association with a first restriction endonuclease and thereby promote the cleavage of the loop portion of the first closed stem-loop oligonucleotide to form the first open stem-loop oligonucleotide.

[0056] Example 8 . The method according to embodiment 7, wherein the restriction endonuclease is a nicking endonuclease that is capable of associating with and cleaving the loop chain of the double-stranded sequence of the first restriction endonuclease.

[0057] Example 9 The method of any one of embodiments 1 to 8, wherein the enzyme has exonuclease activity (e.g., polymerase, exonuclease), and the treating of the reaction mixture comprises:

[0058] The reaction mixture is treated under conditions suitable for:

[0059] - a first target or an amplicon thereof hybridizes to said loop portion of said first closed stem-loop oligonucleotide to form a first double-stranded sequence comprising said first target or an amplicon thereof,

[0060] - a first primer oligonucleotide hybridizing to the first target or an amplicon thereof to form a second double-stranded sequence, the second double-stranded sequence being located upstream (5') relative to the first double-stranded sequence including the first target or an amplicon thereof

[0061] - a first enzyme having exonuclease activity associates with the loop portion of the first closed stem-loop oligonucleotide at or near an end of the first primer oligonucleotide, and

[0062] - catalytic activity of said first enzyme having exonuclease activity, thereby promoting degradation of said loop portion of said first double-stranded sequence comprising said first target or amplicon and forming said first open stem-loop oligonucleotide.

[0063] Example 10 The method of any one of embodiments 1 to 9, wherein the enzyme has exonuclease activity, and the treating of the reaction mixture comprises:

[0064] The reaction mixture is treated under conditions suitable for:

[0065] - a first target or an amplicon thereof hybridizes to said loop portion of said first closed stem-loop oligonucleotide to form a first double-stranded sequence comprising said first target or an amplicon thereof,

[0066] - a first enzyme having exonuclease activity associates with said first double-stranded sequence comprising said first target or an amplicon thereof, and

[0067] - catalytic activity of said first enzyme having exonuclease activity, thereby promoting degradation of said loop portion of said first double-stranded sequence comprising said first target or amplicon and forming said first open stem-loop oligonucleotide.

[0068] Example 11 The method of any one of embodiments 1 to 10, wherein the enzyme comprises a DNA enzyme and / or a ribozyme that requires a first cofactor for catalytic activity, and the treating of the reaction mixture comprises treating the reaction mixture under conditions suitable for:

[0069] - the first cofactor binds to the DNA enzyme and / or the first cofactor binds to the ribozyme so that the DNA enzyme and / or ribozyme exhibit catalytic activity,

[0070] - a DNA enzyme and / or a ribozyme hybridizing to the loop portion of the first closed stem-loop oligonucleotide,

[0071] - the catalytic activity of said DNA enzyme and / or ribozyme, thereby promoting the cleavage of said loop portion of said first closed stem-loop oligonucleotide and forming said first open stem-loop oligonucleotide.

[0072] in:

[0073] The first target is the first cofactor.

[0074] Example 12 . The method according to embodiment 11, wherein the first cofactor is a metal ion (e.g., Mg 2 + 、Mn 2+ , Ca 2+, Pb 2+ ).

[0075] Example 13 The method of any one of embodiments 1 to 12, wherein the treating further comprises treating the reaction mixture under conditions suitable for any one or more of the following:

[0076] - a second MNAzyme binds to the second target or amplicon thereof and the substrate arm of the second MNAzyme hybridises to the loop portion of the second closed stem-loop oligonucleotide, thereby facilitating said cleavage of the loop portion of the second closed stem-loop oligonucleotide by the second MNAzyme forming the second open stem-loop oligonucleotide;

[0077] - hybridizing a second target or an amplicon thereof to said loop portion of said second closed stem-loop oligonucleotide to form a double-stranded sequence for a second restriction endonuclease to associate with said double-stranded sequence and thereby promote said cleavage of said loop portion of said second closed stem-loop oligonucleotide, thereby forming said second open stem-loop oligonucleotide;

[0078] - a second target or an amplicon thereof hybridizes to said loop portion of said second closed stem-loop oligonucleotide to form a second double-stranded sequence comprising said second target or an amplicon thereof,

[0079] a second primer oligonucleotide hybridizing to the second target or an amplicon thereof to form a second double-stranded sequence, the second double-stranded sequence being located upstream (5') relative to the second double-stranded sequence comprising the second target or an amplicon thereof,

[0080] a second enzyme having exonuclease activity associates with the loop portion of the second closed stem-loop oligonucleotide at or near an end of the second primer oligonucleotide, and

[0081] catalytic activity of the second enzyme having exonuclease activity to thereby promote degradation of the loop portion of the second double-stranded sequence comprising the second target or amplicon and form the second open stem-loop oligonucleotide;

[0082] - a second target or an amplicon thereof hybridizes to said loop portion of said second closed stem-loop oligonucleotide to form a second double-stranded sequence comprising said second target or an amplicon thereof,

[0083] a second enzyme having exonuclease activity associates with the second double-stranded sequence comprising the second target or an amplicon thereof, and

[0084] catalytic activity of the second enzyme having exonuclease activity to thereby promote degradation of the loop portion of the second double-stranded sequence comprising the second target or amplicon and form the second open stem-loop oligonucleotide;

[0085] - the second cofactor binds to the DNA enzyme and / or the second cofactor binds to the ribozyme so that the DNA enzyme and / or the ribozyme exhibit catalytic activity,

[0086] A DNA enzyme and / or a ribozyme hybridizes to the loop portion of the second closed stem-loop oligonucleotide,

[0087] The catalytic activity of the DNA enzyme and / or the ribozyme, thereby promoting the cleavage of the loop portion of the second closed stem-loop oligonucleotide and forming the second open stem-loop oligonucleotide.

[0088] in:

[0089] The second target is the second cofactor.

[0090] Example 14 The method of embodiment 13, wherein the second cofactor is a metal ion (e.g., Mg 2 + 、Mn 2+ , Ca 2+ , Pb 2+ ).

[0091] Example 15 . The method according to embodiment 13, wherein the second restriction endonuclease is a nicking endonuclease that is capable of associating with and cleaving the circular strand of the double-stranded sequence comprising the second target or an amplicon thereof.

[0092] Example 16 .The method of embodiment 15, wherein the first restriction endonuclease and the second restriction endonuclease are different types of restriction endonucleases.

[0093] Example 17 .The method of embodiment 15, wherein the first restriction endonuclease and the second restriction endonuclease are the same type of restriction endonuclease.

[0094] Example 18 . The method of embodiment 13, wherein the second target is a nucleic acid sequence or an amplicon thereof, which is capable of hybridizing to the sensor arm of the second MNAzyme, thereby promoting assembly of the second MNAzyme.

[0095] Example 19 The method of embodiment 13, wherein:

[0096] - the second target is an analyte, protein, compound or molecule;

[0097] - the enzyme comprises an enzyme having an aptamer, the aptamer being capable of binding to the second target; and

[0098] - The binding of the second target to the aptamer can cause the enzyme having the aptamer to exhibit catalytic activity.

[0099] Example 20 . The method according to embodiment 19, wherein the enzyme having an aptamer comprises any one or more of the following: an apta-DNA enzyme, an apta-ribozyme, an apta-MNAzyme.

[0100] Example 21 The method of embodiment 13, 19 or 20, wherein:

[0101] - the second target is an analyte, protein, compound or molecule;

[0102] - the reaction mixture further comprises an oligonucleotide sequence capable of hybridising to the sensor arm of the second MNAzyme to thereby facilitate assembly of the second MNAzyme;

[0103] - the second MNAzyme comprises an aptamer sequence capable of binding to the second target; and

[0104] - Binding of the second target to the aptamer sequence of the second MNAzyme is capable of rendering the second MNAzyme catalytically active by facilitating the removal of an inhibitory molecule bound to the aptamer of the second MNAzyme.

[0105] Example 22 . The method of any one of embodiments 1 to 21, wherein the fluorophore of the first closed and open stem-loop oligonucleotides is the same as the fluorophore of the second closed and open stem-loop oligonucleotides.

[0106] Example 23 The method of any one of embodiments 13 to 22, wherein:

[0107] - the reaction mixture comprises the first MNAzyme and the second MNAzyme; and

[0108] - the sequence of the loop portion of the first closed stem-loop oligonucleotide capable of hybridising to the substrate arm of the first MNAzyme is different from the sequence of the loop portion of the second closed stem-loop oligonucleotide capable of hybridising to the substrate arm of the second MNAzyme.

[0109] Example 24 . The method of any one of embodiments 1 to 23, wherein the fluorophores of the first closed and open stem-loop oligonucleotides and the fluorophores of the second closed and open stem-loop oligonucleotides are detectable in a single fluorescence emission channel of a device.

[0110] Example 25The method of any one of embodiments 1 to 24, further comprising determining the presence or absence of a third target or an amplicon thereof in the sample by:

[0111] - contacting a reaction mixture comprising said sample or a derivative thereof with:

[0112] a third closed stem-loop oligonucleotide, wherein the third closed stem-loop oligonucleotide comprises a double-stranded stem portion of hybridized nucleotides linked to a closed single-stranded loop portion of unhybridized nucleotides, wherein:

[0113] the number of hybridizing nucleotides and / or the sequence of hybridizing nucleotides in the stem portion of the third closed stem-loop oligonucleotide is different from the number of hybridizing nucleotides and / or the sequence of hybridizing nucleotides in the stem portion of the first closed stem-loop oligonucleotide and the second closed stem-loop oligonucleotide;

[0114] The double-stranded stem portion includes a fluorophore molecule attached to one strand and a quencher molecule attached to the opposite strand; and

[0115] an enzyme capable of cleaving or degrading the single-stranded loop portion of the third closed stem-loop oligonucleotide only when in contact with the target or an amplicon thereof;

[0116] - treating the reaction mixture under conditions suitable for the enzyme to induce cleavage or degradation of the loop portion of the third closed stem-loop oligonucleotide, thereby generating a third open stem-loop oligonucleotide;

[0117] - detecting the presence or absence of the third oligonucleotide by treating the reaction mixture or a derivative thereof under the following conditions:

[0118] at a third temperature, strand dissociation of the double-stranded stem portion of the third open-loop stem oligonucleotide at the third temperature, thereby promoting spatial separation of the fluorophore and quencher molecule of the stem portion of the third open stem-loop oligonucleotide and providing a third detectable signal,

[0119] in:

[0120] The third temperature is different from the first temperature and the second temperature, and

[0121] Detection of a signal at the third temperature indicates the presence of the third target in the sample, and absence of a signal detected at the third temperature indicates the absence of the third target in the sample.

[0122] Example 26 The method of any one of embodiments 1 to 24, further comprising determining the presence or absence of a third target or an amplicon thereof in the sample by:

[0123] - contacting a reaction mixture comprising said sample or a derivative thereof with:

[0124] a third closed stem-loop oligonucleotide, wherein the third closed stem-loop oligonucleotide comprises a double-stranded stem portion of hybridized nucleotides linked to a closed single-stranded loop portion of unhybridized nucleotides, wherein:

[0125] the number of hybridizing nucleotides and / or the sequence of hybridizing nucleotides in the stem portion of the third closed stem-loop oligonucleotide is the same as the number of hybridizing nucleotides and / or the sequence of hybridizing nucleotides in the stem portion of the first closed stem-loop oligonucleotide or the second closed stem-loop oligonucleotide;

[0126] the double-stranded stem portion comprises a fluorophore molecule attached to one strand and a quencher molecule attached to the opposite strand, wherein the fluorophore molecule attached to the third open stem-loop oligonucleotide emits in a different color region of the visible spectrum than the fluorophore of the first closed stem-loop oligonucleotide and / or the second closed stem-loop oligonucleotide; and

[0127] an enzyme capable of cleaving or degrading the single-stranded loop portion of the third closed stem-loop oligonucleotide only when in contact with the target or an amplicon thereof;

[0128] - treating the reaction mixture under conditions suitable for the enzyme to induce cleavage or degradation of the loop portion of the third closed stem-loop oligonucleotide, thereby generating a third open stem-loop oligonucleotide;

[0129] - detecting the presence or absence of the third oligonucleotide by treating the reaction mixture or a derivative thereof under the following conditions:

[0130] at a third temperature, strand dissociation of the double-stranded stem portion of the third open-loop stem oligonucleotide at the third temperature, thereby promoting spatial separation of the fluorophore and quencher molecule of the stem portion of the third open stem-loop oligonucleotide and providing a third detectable signal,

[0131] in:

[0132] Detection of a signal at the third temperature indicates the presence of the third target in the sample, and absence of a signal detected at the third temperature indicates the absence of the third target in the sample.

[0133] Example 27 .The method according to embodiment 26, wherein the third temperature is different from the first temperature and / or the second temperature.

[0134] Example 28The method of any one of embodiments 25 to 27, wherein the method comprises treating the reaction mixture under conditions suitable for any one or more of the following:

[0135] - a third MNAzyme binds to said third target or amplicon thereof and the substrate arm of said third MNAzyme hybridises to said loop portion of said third closed stem-loop oligonucleotide to thereby facilitate said cleavage of said loop portion of said third closed stem-loop oligonucleotide by said third MNAzyme to form said third open stem-loop oligonucleotide;

[0136] - said third target or an amplicon thereof hybridizes to said loop portion of said third closed stem-loop oligonucleotide to form a double-stranded sequence for a third restriction endonuclease to associate with said double-stranded sequence and thereby promote said cleavage of said loop portion of said third closed stem-loop oligonucleotide, thereby forming said third open stem-loop oligonucleotide;

[0137] - a third target or an amplicon thereof hybridizes to said loop portion of said third closed stem-loop oligonucleotide to form a third double-stranded sequence comprising said third target or an amplicon thereof,

[0138] a third primer oligonucleotide hybridizing to the third target or an amplicon thereof to form a third double-stranded sequence, the third double-stranded sequence being located upstream (5') relative to the third double-stranded sequence including the third target or an amplicon thereof; a third enzyme having exonuclease activity associating with the loop portion of the third closed stem-loop oligonucleotide at or near an end of the third primer oligonucleotide, and

[0139] catalytic activity of the third enzyme having exonuclease activity to thereby promote degradation of the loop portion of the third double-stranded sequence comprising the third target or amplicon and form the third open stem-loop oligonucleotide;

[0140] - a third target or an amplicon thereof hybridizes to said loop portion of said third closed stem-loop oligonucleotide to form a third double-stranded sequence comprising said third target or an amplicon thereof,

[0141] a third enzyme having exonuclease activity that associates with the third double-stranded sequence comprising the third target or an amplicon thereof, and

[0142] catalytic activity of the third enzyme having exonuclease activity to thereby promote degradation of the loop portion of the third double-stranded sequence comprising the third target or amplicon and form the third open stem-loop oligonucleotide;

[0143] - the third cofactor binds to the DNA enzyme and / or the third cofactor binds to the ribozyme so that the DNA enzyme and / or the ribozyme exhibit catalytic activity,

[0144] A DNA enzyme and / or a ribozyme hybridizes to said loop portion of the third closed stem-loop oligonucleotide,

[0145] The catalytic activity of the DNA enzyme and / or the ribozyme, thereby promoting the cleavage of the loop portion of the third closed stem-loop oligonucleotide and forming the third open stem-loop oligonucleotide.

[0146] in:

[0147] The third target is the third cofactor.

[0148] Example 29 The method according to embodiment 28, wherein the cofactor is a metal ion (e.g., Mg 2+ 、Mn 2 + , Ca 2+ , Pb 2+ ).

[0149] Example 30 .A method according to embodiment 28, wherein the restriction endonuclease is a nicking endonuclease that is capable of associating with and cleaving the circular strand of the double-stranded sequence comprising the third target or an amplicon thereof.

[0150] Example 31 . The method of embodiment 28, wherein the third target is a nucleic acid sequence or an amplicon thereof, which is capable of hybridizing to the sensor arm of the third MNAzyme, thereby promoting assembly of the third MNAzyme.

[0151] Example 32 The method of embodiment 28, wherein:

[0152] - the target is an analyte, protein, compound or molecule;

[0153] - the enzyme comprises an enzyme having an aptamer, the aptamer being capable of binding to the third target; and

[0154] - The binding of the third target to the aptamer can cause the enzyme with the aptamer to exhibit catalytic activity

[0155] Example 33 . The method according to embodiment 32, wherein the enzyme having an aptamer comprises any one or more of the following: an apta-DNA enzyme, an apta-ribozyme, an apta-MNAzyme.

[0156] Example 34 The method of embodiment 28, wherein:

[0157] - the third target is an analyte, protein, compound or molecule;

[0158] - the reaction mixture further comprises an oligonucleotide sequence capable of hybridising to the sensor arm of the third MNAzyme to thereby facilitate assembly of the third MNAzyme;

[0159] - said third MNAzyme comprises an aptamer sequence capable of binding to said third target; and

[0160] - Binding of the third target to the aptamer sequence of the third MNAzyme is capable of rendering the third MNAzyme catalytically active by facilitating the removal of an inhibitory molecule bound to the aptamer of the third MNAzyme.

[0161] Example 35 The method of any one of embodiments 28 or 30 to 34, wherein:

[0162] - the reaction mixture comprises the third MNAzyme and either the first MNAzyme or the second MNAzyme; and

[0163] - the sequence of the loop portion of the third closed stem-loop oligonucleotide capable of hybridising to the substrate arm of the third MNAzyme is different from:

[0164] the sequence of the loop portion of the first closed stem-loop oligonucleotide capable of hybridizing to the substrate arm of the first MNAzyme, and / or

[0165] The sequence of the loop portion of the second closed stem-loop oligonucleotide is capable of hybridising to the substrate arm of the first MNAzyme.

[0166] Example 36 The method of embodiment 25 or any one of embodiments 28 to 35, wherein:

[0167] - the number of hybridizing nucleotides and / or the sequence of hybridizing nucleotides in the stem portion of the third closed stem-loop oligonucleotide is different from the number of hybridizing nucleotides and / or the sequence of hybridizing nucleotides in the stem portion of the first closed stem-loop oligonucleotide and the second closed stem-loop oligonucleotide;

[0168] - the third temperature is different from the first temperature and the second temperature; and

[0169] - the fluorophores of the third closed stem-loop oligonucleotide and the third open stem-loop oligonucleotide emit in the same color region of the visible spectrum as the fluorophores of the first closed stem-loop oligonucleotide and the second closed stem-loop oligonucleotide and the first open stem-loop oligonucleotide and the second open stem-loop oligonucleotide.

[0170] Example 37. The method of embodiment 25 or any one of embodiments 28 to 36, wherein the fluorophore of the third closed and open stem-loop oligonucleotide is the same as the fluorophore of the first and / or second closed and open stem-loop oligonucleotide.

[0171] Example 38 .A method according to embodiment 37, wherein the fluorophore of the third closed and open stem-loop oligonucleotides and the fluorophore of the first closed and open stem-loop oligonucleotides and / or the fluorophore of the second closed and open oligonucleotides can be detected using the same emission channel of the device.

[0172] Example 39 The method of any one of embodiments 25 to 35 or 36, wherein:

[0173] - the fluorophores of the first closed stem-loop oligonucleotide and the first open stem-loop oligonucleotide emit in the same color region of the visible spectrum as the fluorophores of the second closed stem-loop oligonucleotide and the second open stem-loop oligonucleotide; and

[0174] - the fluorophores of the third closed stem-loop oligonucleotide and the open stem-loop oligonucleotide emit in a different color region of the visible spectrum than the fluorophores of the first closed stem-loop oligonucleotide and the second closed stem-loop oligonucleotide and the first open stem-loop oligonucleotide and the second open stem-loop oligonucleotide.

[0175] Example 40 .The method according to any one of embodiments 25 to 39, wherein the third temperature differs from the first temperature and / or the second temperature by more than: 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C.

[0176] Example 41 . The method of any one of embodiments 1 to 40, wherein melting curve analysis is used for any said detection of a signal or any said failure to detect a signal.

[0177] Example 42.The method according to any one of embodiments 1 to 41, wherein the first temperature differs from the second temperature by more than: 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C.

[0178] Example 43 .A method according to one of embodiments 1 to 42, wherein any of the amplicons are produced by any one or more of the following: polymerase chain reaction (PCR), strand displacement amplification (SDA), helicase-dependent amplification (HDA), recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), rolling circle amplification (RCA), transcription-mediated amplification (TMA), self-sustained sequence replication (3SR), nucleic acid sequence-based amplification (NASBA) and / or reverse transcription polymerase chain reaction (RT-PCR).

[0179] Example 44 . The method according to one of embodiments 1 to 43, wherein the sample is a biological sample obtained from a subject.

[0180] Example 45 . The method according to one of embodiments 1 to 43, wherein the method is performed in vitro.

[0181] Example 46 . The method according to one of embodiments 1 to 43, wherein the method is performed ex vivo.

[0182] Example 47 A composition comprising:

[0183] a first closed stem-loop oligonucleotide and a second closed stem-loop oligonucleotide, wherein each of the closed stem-loop oligonucleotides comprises a double-stranded stem portion of hybridized nucleotides joined to a single-stranded loop portion of unhybridized nucleotides, wherein:

[0184] The first closed stem-loop oligonucleotide and the second closed stem-loop oligonucleotide differ in the number of hybridizing nucleotides and / or the sequence of hybridizing nucleotides in the stem portion, and

[0185] Each of the double-stranded stem portions comprises a fluorophore molecule attached to one strand and a quencher molecule attached to the opposite strand,

[0186] wherein the fluorophore molecules emit in the same color region of the visible spectrum and the melting temperature (Tm) of the double-stranded stem portion of the first closed stem-loop oligonucleotide is different from the Tm of the double-stranded stem portion of the second closed stem-loop oligonucleotide.

[0187] Example 48 . The composition of embodiment 47, wherein the single-stranded loop portion of the first closed stem-loop oligonucleotide is different in sequence from the single-stranded loop portion of the second closed stem-loop oligonucleotide.

[0188] Example 49 The composition of embodiment 47 or embodiment 48, further comprising:

[0189] a first MNAzyme comprising a substrate arm capable of hybridising to the closed single stranded loop portion of the first closed stem-loop oligonucleotide; and

[0190] A second MNAzyme comprising a substrate arm capable of hybridising to the single stranded loop portion of the second closed stem-loop oligonucleotide.

[0191] Example 50 A composition according to embodiment 49, wherein:

[0192] the substrate arm of the first MNAzyme hybridizes to the single stranded loop portion of the first closed stem-loop oligonucleotide; and

[0193] The substrate arm of the second MNAzyme hybridizes to the single stranded loop portion of the second closed stem-loop oligonucleotide.

[0194] Example 51 A composition according to embodiment 50, wherein:

[0195] the first MNAzyme and / or the second MNAzyme comprises an aptamer sequence that binds to a target analyte, protein, compound or molecule and a sensor arm that hybridizes to an oligonucleotide sequence; and

[0196] The first MNAzyme is designed to detect a different target than the second MNAzyme.

[0197] Example 52 A composition according to embodiment 50, wherein:

[0198] The first MNAzyme and / or the second MNAzyme comprises a sensor arm that hybridizes to a target sequence, and the first MNAzyme is designed to detect a different target than the second MNAzyme.

[0199] Example 53 The composition of any one of embodiments 47 to 52, further comprising:

[0200] a first oligonucleotide sequence that hybridizes to the single-stranded loop portion of the first closed stem-loop oligonucleotide that is different in sequence from the single-stranded loop portion of the second closed stem-loop oligonucleotide, thereby forming a double-stranded sequence including the first oligonucleotide;

[0201] a second oligonucleotide sequence that hybridizes to the single-stranded loop portion of the second closed stem-loop oligonucleotide, the second oligonucleotide sequence being different from the sequence of the single-stranded loop portion of the first closed stem-loop oligonucleotide, thereby forming a double-stranded sequence including the second oligonucleotide;

[0202] a restriction endonuclease that associates with and is capable of cleaving the double-stranded sequence comprising the first oligonucleotide; and / or

[0203] A restriction endonuclease that associates with and is capable of cleaving the double-stranded sequence comprising the second oligonucleotide.

[0204] Example 54 The composition of any one of embodiments 47 to 53, further comprising:

[0205] a first double-stranded sequence comprising a first target oligonucleotide sequence that hybridizes to the single-stranded loop portion of the first closed stem-loop oligonucleotide having a sequence different from that of the single-stranded loop portion of the second closed stem-loop oligonucleotide, thereby forming a first double-stranded sequence comprising the first target oligonucleotide;

[0206] a second double-stranded sequence comprising a second target oligonucleotide sequence that hybridizes to the single-stranded loop portion of the second closed stem-loop oligonucleotide having a sequence different from that of the single-stranded loop portion of the first closed stem-loop oligonucleotide, thereby forming a double-stranded sequence comprising the second target oligonucleotide;

[0207] a first primer oligonucleotide that hybridizes to the first target oligonucleotide upstream (5') relative to the first double-stranded sequence, and a first enzyme having exonuclease activity (e.g., a polymerase, an exonuclease) that associates with the loop portion of the first closed stem-loop oligonucleotide at or near an end of the first primer oligonucleotide; and / or

[0208] a second primer oligonucleotide that hybridizes to the second target oligonucleotide upstream (5') relative to the second double-stranded sequence, and a second enzyme with exonuclease activity (e.g., a polymerase, an exonuclease) that associates with the loop portion of the second closed stem-loop oligonucleotide at or near an end of the second primer oligonucleotide.

[0209] Example 55 The composition of any one of embodiments 47 to 54, further comprising:

[0210] a first double-stranded sequence comprising a first target oligonucleotide sequence that hybridizes to the single-stranded loop portion of the closed first stem-loop oligonucleotide having a sequence different from that of the single-stranded loop portion of the second closed stem-loop oligonucleotide, thereby forming a first double-stranded sequence comprising the first target oligonucleotide;

[0211] a second double-stranded sequence comprising a second target oligonucleotide sequence that hybridizes to the single-stranded loop portion of the second closed stem-loop oligonucleotide having a sequence different from that of the single-stranded loop portion of the first closed stem-loop oligonucleotide, thereby forming a second double-stranded sequence comprising the second target oligonucleotide;

[0212] A first enzyme having exonuclease activity associates with the first double-stranded sequence, and a second enzyme having exonuclease activity associates with the second double-stranded sequence.

[0213] Example 56 The composition of any one of embodiments 47 to 55, further comprising:

[0214] a first DNA enzyme and / or a first ribozyme that hybridizes to a loop portion of the first closed stem-loop oligonucleotide that has a sequence different from that of the single-stranded loop portion of the second closed stem-loop oligonucleotide,

[0215] a second DNA enzyme and / or a second ribozyme that hybridizes to the loop portion of the second closed stem-loop oligonucleotide having a sequence different from that of the single-stranded loop portion of the first closed stem-loop oligonucleotide,

[0216] a cofactor for each of the DNA enzymes, the cofactor being capable of causing each of the DNA enzymes to exhibit catalytic activity and thereby inducing cleavage activity of any of the DNA enzymes hybridized to the loop portion.

[0217] a cofactor for each of said ribozymes, said cofactor being capable of rendering each of said ribozymes catalytically active and thereby inducing the cleavage activity of any of said ribozymes hybridized to said loop portion.

[0218] Embodiment 57. The composition of any one of embodiments 47 to 56, further comprising:

[0219] a third closed stem-loop oligonucleotide, wherein the third closed stem-loop oligonucleotide comprises a double-stranded stem portion of hybridized nucleotides linked to a single-stranded loop portion of unhybridized nucleotides, wherein:

[0220] The number of hybridizing nucleotides and / or the sequence of hybridizing nucleotides in the stem portion of the third closed stem-loop oligonucleotide is different from the number of hybridizing nucleotides and / or the sequence of hybridizing nucleotides in the stem portion of the first closed stem-loop oligonucleotide and the second closed stem-loop oligonucleotide, and

[0221] The double-stranded stem portion of the third closed stem-loop oligonucleotide comprises a fluorophore molecule attached to one strand and a quencher molecule attached to the opposite strand, wherein the fluorophore molecule of the third closed stem-loop oligonucleotide emits in a different color region of the visible spectrum than the fluorophore molecules of the first closed stem-loop oligonucleotide and the second closed stem-loop oligonucleotide.

[0222] Embodiment 58. The composition of any one of embodiments 47 to 56, further comprising:

[0223] a third closed stem-loop oligonucleotide, wherein the third closed stem-loop oligonucleotide comprises a double-stranded stem portion of hybridized nucleotides linked to a single-stranded loop portion of unhybridized nucleotides, wherein:

[0224] The number of hybridizing nucleotides and / or the sequence of hybridizing nucleotides in the stem portion of the third closed stem-loop oligonucleotide is different from the number of hybridizing nucleotides and / or the sequence of hybridizing nucleotides in the stem portion of the first closed stem-loop oligonucleotide and the second closed stem-loop oligonucleotide, and

[0225] The double-stranded stem portion of the third closed stem-loop oligonucleotide comprises a fluorophore molecule attached to one strand and a quencher molecule attached to the opposite strand, wherein the fluorophore molecule of the third closed stem-loop oligonucleotide emits in the same color region of the visible spectrum as the fluorophore molecules of the first closed stem-loop oligonucleotide and the second closed stem-loop oligonucleotide.

[0226] Embodiment 59. The composition of any one of embodiments 47 to 56, further comprising:

[0227] a third closed stem-loop oligonucleotide, wherein the third closed stem-loop oligonucleotide comprises a double-stranded stem portion of hybridized nucleotides linked to a single-stranded loop portion of unhybridized nucleotides, wherein:

[0228] The number of hybridizing nucleotides and / or the sequence of hybridizing nucleotides in the stem portion of the third closed stem-loop oligonucleotide is the same as the number of hybridizing nucleotides and / or the sequence of hybridizing nucleotides in the stem portion of the first closed stem-loop oligonucleotide or the second closed stem-loop oligonucleotide, and

[0229] The double-stranded stem portion of the third closed stem-loop oligonucleotide comprises a fluorophore molecule attached to one strand and a quencher molecule attached to the opposite strand, wherein the fluorophore molecule of the third closed stem-loop oligonucleotide emits in a different color region of the visible spectrum than the fluorophore molecules of the first closed stem-loop oligonucleotide and the second closed stem-loop oligonucleotide.

[0230] The present invention also relates to the following examples 1-81:

[0231] Example 1. A method for determining the presence or absence of a first target and a second target in a sample, the method comprising:

[0232] (a) preparing a reaction mixture by contacting said sample, or a derivative thereof, presumably comprising said first target and / or said second target, or amplicons thereof, with:

[0233] a first closed stem-loop oligonucleotide and a second closed stem-loop oligonucleotide, wherein each of the closed stem-loop oligonucleotides comprises a double-stranded stem portion of hybridized nucleotides joined to a closed single-stranded loop portion of unhybridized nucleotides, wherein:

[0234] The first closed stem-loop oligonucleotide and the second closed stem-loop oligonucleotide differ in the number of hybridizing nucleotides and / or the sequence of hybridizing nucleotides in the stem portion, and

[0235] an enzyme capable of cleaving or degrading the single-stranded loop portions of the first and second closed stem-loop oligonucleotides only when in contact with the target or an amplicon thereof;

[0236] (b) treating the reaction mixture:

[0237] - under conditions suitable for said enzyme to induce cleavage or degradation of said loop portions of said first closed stem-loop oligonucleotide and said second closed stem-loop oligonucleotide to thereby produce first and second open stem-loop oligonucleotides;

[0238] - at a first temperature, at or above which the strands of the double-stranded stem portion of the first open stem-loop oligonucleotide dissociate to thereby promote spatial separation of the fluorophore molecule and the quencher molecule of the stem portion of the first open stem-loop oligonucleotide and provide a first detectable fluorescent signal, and

[0239] - at a second temperature, at or above which the strands of the double-stranded stem portion of the second open stem-loop oligonucleotide dissociate to thereby facilitate spatial separation of the fluorophore molecule and the quencher molecule of the stem portion of the second open stem-loop oligonucleotide and provide a second detectable fluorescent signal;

[0240] in:

[0241] The first temperature is lower than the second temperature, and

[0242] The fluorophore of the first open stem-loop oligonucleotide and the fluorophore of the second open stem-loop oligonucleotide emit in the same color region of the visible spectrum; and

[0243] (c) detecting the levels of the first and second fluorescent signals at one or more temperatures including or consisting of a temperature equal to or greater than the second temperature, thereby determining the presence or absence of the target in the sample.

[0244] Example 2. The method of embodiment 1, wherein the enzyme comprises a multicomponent nucleic acid enzyme (MNAzyme), and the treating of the reaction mixture comprises treating the reaction mixture under conditions suitable for:

[0245] A first multicomponent nucleic acid enzyme (MNAzyme) binds to the first target or an amplicon thereof and the substrate arm of the first MNAzyme hybridizes to the loop portion of the first closed stem-loop oligonucleotide to thereby promote said cleavage of the loop portion of the first closed stem-loop oligonucleotide by the first MNAzyme to form the first open stem-loop oligonucleotide.

[0246] Example 3 . The method of embodiment 2, wherein the first target is a nucleic acid sequence or an amplicon thereof, which is capable of hybridizing to the sensor arm of the first MNAzyme, thereby promoting assembly of the first MNAzyme.

[0247] Example 4 The method of embodiment 1, wherein:

[0248] - the first target is an analyte, protein, compound or molecule;

[0249] - the enzyme comprises an enzyme having an aptamer, the aptamer being capable of binding to the first target; and

[0250] - The binding of the first target to the aptamer enables the enzyme having the aptamer to exhibit catalytic activity.

[0251] Example 5. The method according to embodiment 4, wherein the enzyme having an aptamer comprises any one or more of the following: an apta-DNA enzyme, an apta-ribozyme, an apta-MNAzyme.

[0252] Example 6 The method of any one of embodiments 2, 4 or 5, wherein:

[0253] - the first target is an analyte, protein, compound or molecule;

[0254] - the reaction mixture further comprises an oligonucleotide sequence capable of hybridizing to the sensor arm of the first MNAzyme to thereby facilitate assembly of the first MNAzyme;

[0255] - said first MNAzyme comprises an aptamer sequence capable of binding to said first target; and

[0256] - Binding of the target to the aptamer enables the first MNAzyme to exhibit catalytic activity.

[0257] Example 7 . The method of any one of embodiments 1 to 6, wherein said closed stem-loop oligonucleotide does not hybridize to said target or an amplicon thereof during said cleavage or degradation by said enzyme.

[0258] Example 8 The method of any one of embodiments 1 to 6, wherein the enzyme comprises a restriction endonuclease, and the treating of the reaction mixture comprises:

[0259] The reaction mixture is treated under conditions suitable for hybridization of the first target or an amplicon thereof to the loop portion of the first closed stem-loop oligonucleotide to form a double-stranded sequence for association with a first restriction endonuclease and thereby promote the cleavage of the loop portion of the first closed stem-loop oligonucleotide to form the first open stem-loop oligonucleotide.

[0260] Example 9 . The method according to embodiment 8, wherein the restriction endonuclease is a nicking endonuclease that is capable of associating with and cleaving the loop strand of the double-stranded sequence of the first restriction endonuclease.

[0261] Example 10 The method of any one of embodiments 1 to 6, wherein the enzyme has exonuclease activity (e.g., polymerase, exonuclease), and the treating of the reaction mixture comprises:

[0262] The reaction mixture is treated under conditions suitable for:

[0263] - a first target or an amplicon thereof hybridizes to said loop portion of said first closed stem-loop oligonucleotide to form a first double-stranded sequence comprising said first target or an amplicon thereof,

[0264] - a first primer oligonucleotide hybridizing to the first target or an amplicon thereof to form a second double-stranded sequence, the second double-stranded sequence being located upstream (5') relative to the first double-stranded sequence including the first target or an amplicon thereof

[0265] - a first enzyme having exonuclease activity associates with said loop portion of said first closed stem-loop oligonucleotide at or near an end of said first primer oligonucleotide, and

[0266] - catalytic activity of said first enzyme having exonuclease activity, to thereby promote degradation of the loop portion of said first double-stranded sequence comprising said first target or amplicon and to form said first open stem-loop oligonucleotide.

[0267] Example 11 The method of any one of embodiments 1 to 6, wherein the enzyme has exonuclease activity, and the treating of the reaction mixture comprises:

[0268] The reaction mixture is treated under conditions suitable for:

[0269] - a first target or an amplicon thereof hybridizes to said loop portion of said first closed stem-loop oligonucleotide to form a first double-stranded sequence comprising said first target or an amplicon thereof,

[0270] - a first enzyme having exonuclease activity associates with said first double-stranded sequence comprising said first target or an amplicon thereof, and

[0271] - catalytic activity of said first enzyme having exonuclease activity, to thereby promote degradation of the loop portion of said first double-stranded sequence comprising said first target or amplicon and to form said first open stem-loop oligonucleotide.

[0272] Example 12. The method of any one of embodiments 1 to 11, wherein said enzyme comprises a DNA enzyme and / or a ribozyme that requires a first cofactor for catalytic activity, and said treating of said reaction mixture comprises treating said reaction mixture under conditions suitable for:

[0273] - the first cofactor binds to the DNA enzyme and / or the first cofactor binds to the ribozyme so that the DNA enzyme and / or the ribozyme exhibit catalytic activity,

[0274] - a DNA enzyme and / or a ribozyme hybridizing to said loop portion of the first closed stem-loop oligonucleotide,

[0275] - the catalytic activity of said DNA enzyme and / or said ribozyme, to thereby promote the cleavage of said loop portion of said first closed stem-loop oligonucleotide and to form said first open stem-loop oligonucleotide.

[0276] in:

[0277] The first target is the first cofactor.

[0278] Example 13 The method of embodiment 12, wherein the first cofactor is a metal ion (e.g., Mg 2 + 、Mn 2+ , Ca 2+ , Pb 2+ ).

[0279] Example 14 The method of any one of embodiments 1 to 13, wherein the treating further comprises treating the reaction mixture under conditions suitable for any one or more of the following:

[0280] - a second MNAzyme binds to the second target or amplicon thereof and the substrate arm of the second MNAzyme hybridizes to the loop portion of the second closed stem-loop oligonucleotide to thereby facilitate said cleavage of the loop portion of the second closed stem-loop oligonucleotide by the second MNAzyme to form the second open stem-loop oligonucleotide;

[0281] - hybridizing a second target or an amplicon thereof to said loop portion of said second closed stem-loop oligonucleotide to form a double-stranded sequence for a second restriction endonuclease to associate with said double-stranded sequence and thereby promote said cleavage of said loop portion of said second closed stem-loop oligonucleotide, thereby forming said second open stem-loop oligonucleotide;

[0282] - a second target or an amplicon thereof hybridizes to said loop portion of said second closed stem-loop oligonucleotide to form a second double-stranded sequence comprising said second target or an amplicon thereof,

[0283] a second primer oligonucleotide hybridizing to the second target or an amplicon thereof to form a second double-stranded sequence, the second double-stranded sequence being located upstream (5') relative to the second double-stranded sequence comprising the second target or an amplicon thereof,

[0284] a second enzyme having exonuclease activity associates with the loop portion of the second closed stem-loop oligonucleotide at or near an end of the second primer oligonucleotide, and

[0285] catalytic activity of the second enzyme having exonuclease activity to thereby promote degradation of the loop portion of the second double-stranded sequence comprising the second target or amplicon and form the second open stem-loop oligonucleotide;

[0286] - a second target or an amplicon thereof hybridizes to said loop portion of said second closed stem-loop oligonucleotide to form a second double-stranded sequence comprising said second target or an amplicon thereof,

[0287] a second enzyme having exonuclease activity associates with the second double-stranded sequence comprising the second target or an amplicon thereof, and

[0288] catalytic activity of the second enzyme having exonuclease activity to thereby promote degradation of the loop portion of the second double-stranded sequence comprising the second target or amplicon and form the second open stem-loop oligonucleotide;

[0289] - the second cofactor binds to the DNA enzyme and / or the second cofactor binds to the ribozyme so that the DNA enzyme and / or the ribozyme exhibit catalytic activity,

[0290] A DNA enzyme and / or a ribozyme hybridizes to the loop portion of the second closed stem-loop oligonucleotide,

[0291] The catalytic activity of the DNA enzyme and / or the ribozyme, thereby promoting the cleavage of the loop portion of the second closed stem-loop oligonucleotide and forming the second open stem-loop oligonucleotide.

[0292] in:

[0293] The second target is the second cofactor.

[0294] Example 15 The method of embodiment 14, wherein the second cofactor is a metal ion (e.g., Mg 2 + 、Mn 2+ , Ca 2+ , Pb 2+ ).

[0295] Example 16 . The method according to embodiment 14, wherein the second restriction endonuclease is a nicking endonuclease that is capable of associating with and cleaving the circular strand of the double-stranded sequence comprising the second target or an amplicon thereof.

[0296] Example 17 .The method of embodiment 16, wherein the first and second restriction endonucleases are different types of restriction endonucleases.

[0297] Example 18.The method of embodiment 16, wherein the first and second restriction endonucleases are the same type of restriction endonucleases.

[0298] Example 19 . The method of embodiment 14, wherein the second target is a nucleic acid sequence or an amplicon thereof, which is capable of hybridizing to the sensor arm of the second MNAzyme, thereby promoting assembly of the second MNAzyme.

[0299] Example 20 The method of embodiment 14, wherein:

[0300] - the second target is an analyte, protein, compound or molecule;

[0301] - the enzyme comprises an enzyme having an aptamer, the aptamer being capable of binding to the second target; and

[0302] - The binding of the second target to the aptamer can cause the enzyme having the aptamer to exhibit catalytic activity.

[0303] Example 21 . The method according to embodiment 20, wherein the enzyme having an aptamer comprises any one or more of the following: an apta-DNA enzyme, an apta-ribozyme, an apta-MNAzyme.

[0304] Example 22 The method of embodiment 14, 20, or 21, wherein:

[0305] - the second target is an analyte, protein, compound or molecule;

[0306] - the reaction mixture further comprises an oligonucleotide sequence capable of hybridising to the sensor arm of the second MNAzyme to thereby facilitate assembly of the second MNAzyme;

[0307] - the second MNAzyme comprises an aptamer sequence capable of binding to the second target; and

[0308] - Binding of the second target to the aptamer sequence of the second MNAzyme is capable of rendering the second MNAzyme catalytically active by facilitating the removal of an inhibitory molecule bound to the aptamer of the second MNAzyme.

[0309] Example 23 . The method of any one of embodiments 1 to 22, wherein the fluorophore of the first closed and open stem-loop oligonucleotides is the same as the fluorophore of the second closed and open stem-loop oligonucleotides.

[0310] Example 24 The method of any one of embodiments 14 to 23, wherein:

[0311] - the reaction mixture comprises the first MNAzyme and the second MNAzyme; and

[0312] - the sequence of the loop portion of the first closed stem-loop oligonucleotide capable of hybridising to the substrate arm of the first MNAzyme is different from the sequence of the loop portion of the second closed stem-loop oligonucleotide capable of hybridising to the substrate arm of the second MNAzyme.

[0313] Example 25 . The method of any one of embodiments 1 to 24, wherein the fluorophore of the first closed and open stem-loop oligonucleotides and the fluorophore of the second closed and open stem-loop oligonucleotides are detectable in a single fluorescence emission channel of a device.

[0314] Example 26 . The method of any one of embodiments 1 to 25, wherein said enzyme does not induce cleavage or degradation of any of said targets or amplicons thereof.

[0315] Example 27 . The method of any one of embodiments 1 to 26, wherein the first and second closed stem-loop oligonucleotides each consist of the double-stranded stem portion and the single-stranded loop portion.

[0316] Example 28 The method of any one of embodiments 1 to 27, further comprising determining the presence or absence of a third target or an amplicon thereof in the sample by:

[0317] (d) contacting said reaction mixture comprising said sample or a derivative thereof with:

[0318] a third closed stem-loop oligonucleotide, wherein the third closed stem-loop oligonucleotide comprises a double-stranded stem portion of hybridized nucleotides linked to a closed single-stranded loop portion of unhybridized nucleotides, wherein:

[0319] the number of hybridizing nucleotides and / or the sequence of hybridizing nucleotides in the stem portion of the third closed stem-loop oligonucleotide is different from the number of hybridizing nucleotides and / or the sequence of hybridizing nucleotides in the stem portion of the first closed stem-loop oligonucleotide and the second closed stem-loop oligonucleotide;

[0320] The double-stranded stem portion includes a fluorophore molecule attached to one strand and a quencher molecule attached to the opposite strand; and

[0321] an enzyme capable of cleaving or degrading the single-stranded loop portion of the third closed stem-loop oligonucleotide only when in contact with the target or an amplicon thereof;

[0322] (e) treating the reaction mixture:

[0323] - under conditions suitable for said enzyme to induce cleavage or degradation of said loop portion of said third closed stem-loop oligonucleotide to thereby produce a third open stem-loop oligonucleotide;

[0324] - at a third temperature, at or above the third temperature, the strands of the double-stranded stem portion of the third open stem-loop oligonucleotide dissociate to thereby promote spatial separation of the fluorophore molecule and the quencher molecule of the stem portion of the third open stem-loop oligonucleotide and provide a third detectable fluorescent signal;

[0325] in:

[0326] The third temperature is higher than the first temperature and the second temperature, and

[0327] (f) detecting the levels of the first fluorescent signal, the second fluorescent signal, and the third fluorescent signal at one or more temperatures including or consisting of a temperature equal to or higher than the third temperature to thereby determine the presence or absence of the target in the sample.

[0328] Example 29 The method of any one of embodiments 1 to 27, further comprising determining the presence or absence of a third target or an amplicon thereof in the sample by:

[0329] (d) contacting said reaction mixture comprising said sample or a derivative thereof with:

[0330] a third closed stem-loop oligonucleotide, wherein the third closed stem-loop oligonucleotide comprises a double-stranded stem portion of hybridized nucleotides linked to a closed single-stranded loop portion of unhybridized nucleotides, wherein:

[0331] the number of hybridizing nucleotides and / or the sequence of hybridizing nucleotides in the stem portion of the third closed stem-loop oligonucleotide is the same as the number of hybridizing nucleotides and / or the sequence of hybridizing nucleotides in the stem portion of the first closed stem-loop oligonucleotide or the second closed stem-loop oligonucleotide;

[0332] the double-stranded stem portion comprises a fluorophore molecule attached to one strand and a quencher molecule attached to the opposite strand, wherein the fluorophore molecule attached to the third open stem-loop oligonucleotide emits in a different color region of the visible spectrum than the fluorophore of the first closed stem-loop oligonucleotide and / or the second closed stem-loop oligonucleotide; and

[0333] an enzyme capable of cleaving or degrading the single-stranded loop portion of the third closed stem-loop oligonucleotide only when in contact with the target or an amplicon thereof;

[0334] (e) treating the reaction mixture:

[0335] - under conditions suitable for said enzyme to induce cleavage or degradation of said loop portion of said third closed stem-loop oligonucleotide to thereby produce a third open stem-loop oligonucleotide;

[0336] - at a third temperature, at which the strands of the double-stranded stem portion of the third open stem-loop oligonucleotide dissociate to thereby promote spatial separation of the fluorophore molecule and the quencher molecule of the stem portion of the third open stem-loop oligonucleotide and provide a third detectable fluorescent signal;

[0337] (f) detecting the levels of the first fluorescent signal, the second fluorescent signal, and the third fluorescent signal at one or more temperatures including or consisting of a temperature equal to or higher than the third temperature to thereby determine the presence or absence of the target in the sample.

[0338] Example 30 .The method according to embodiment 29, wherein the third temperature is different from the first and / or second temperature.

[0339] Example 31 The method of any one of embodiments 28 to 30, wherein the method comprises treating the reaction mixture under conditions suitable for any one or more of the following:

[0340] - a third MNAzyme binds to said third target or amplicon thereof and the substrate arm of said third MNAzyme hybridises to said loop portion of said third closed stem-loop oligonucleotide to thereby facilitate said cleavage of said loop portion of said third closed stem-loop oligonucleotide by said third MNAzyme to form said third open stem-loop oligonucleotide;

[0341] - said third target or an amplicon thereof hybridizes to said loop portion of said third closed stem-loop oligonucleotide to form a double-stranded sequence for a third restriction endonuclease to associate with said double-stranded sequence and thereby promote said cleavage of said loop portion of said third closed stem-loop oligonucleotide, thereby forming said third open stem-loop oligonucleotide;

[0342] - a third target or an amplicon thereof hybridizes to said loop portion of said third closed stem-loop oligonucleotide to form a third double-stranded sequence comprising said third target or an amplicon thereof,

[0343] a third primer oligonucleotide hybridizing to the third target or an amplicon thereof to form a third double-stranded sequence, the third double-stranded sequence being located upstream (5') relative to the third double-stranded sequence including the third target or an amplicon thereof; a third enzyme having exonuclease activity associating with the loop portion of the third closed stem-loop oligonucleotide at or near an end of the third primer oligonucleotide, and

[0344] catalytic activity of the third enzyme having exonuclease activity to thereby promote degradation of the loop portion of the third double-stranded sequence comprising the third target or amplicon and form the third open stem-loop oligonucleotide;

[0345] - a third target or an amplicon thereof hybridizes to said loop portion of said third closed stem-loop oligonucleotide to form a third double-stranded sequence comprising said third target or an amplicon thereof,

[0346] a third enzyme having exonuclease activity that associates with the third double-stranded sequence comprising the third target or an amplicon thereof, and

[0347] catalytic activity of the third enzyme having exonuclease activity to thereby promote degradation of the loop portion of the third double-stranded sequence comprising the third target or amplicon and form the third open stem-loop oligonucleotide;

[0348] - the third cofactor binds to the DNA enzyme and / or the third cofactor binds to the ribozyme so that the DNA enzyme and / or the ribozyme exhibit catalytic activity,

[0349] A DNA enzyme and / or a ribozyme hybridizes to said loop portion of the third closed stem-loop oligonucleotide,

[0350] The catalytic activity of the DNA enzyme and / or the ribozyme, thereby promoting the cleavage of the loop portion of the third closed stem-loop oligonucleotide and forming the third open stem-loop oligonucleotide.

[0351] in:

[0352] The third target is the third cofactor.

[0353] Example 32 The method according to embodiment 31, wherein the cofactor is a metal ion (e.g., Mg 2+ 、Mn 2 + , Ca 2+ , Pb 2+ ).

[0354] Example 33 .A method according to embodiment 31, wherein the restriction endonuclease is a nicking endonuclease that is capable of associating with and cleaving the circular strand of the double-stranded sequence comprising the third target or an amplicon thereof.

[0355] Example 34 . The method of embodiment 31 wherein the third target is a nucleic acid sequence or an amplicon thereof that is capable of hybridizing to the sensor arm of the third MNAzyme, thereby promoting assembly of the third MNAzyme.

[0356] Example 35 The method of embodiment 31, wherein:

[0357] - the target is an analyte, protein, compound or molecule;

[0358] - the enzyme comprises an enzyme having an aptamer, the aptamer being capable of binding to the third target; and

[0359] - The binding of the third target to the aptamer can cause the enzyme with the aptamer to exhibit catalytic activity

[0360] Example 36 .The method according to embodiment 35, wherein the enzyme with an aptamer comprises any one or more of the following: an apta-DNA enzyme, an apta-ribozyme, an apta-MNAzyme.

[0361] Example 37 The method of embodiment 31, wherein:

[0362] - the third target is an analyte, protein, compound or molecule;

[0363] - the reaction mixture further comprises an oligonucleotide sequence capable of hybridising to the sensor arm of the third MNAzyme to thereby facilitate assembly of the third MNAzyme;

[0364] - said third MNAzyme comprises an aptamer sequence capable of binding to said third target; and

[0365] - Binding of the third target to the aptamer sequence of the third MNAzyme is capable of rendering the third MNAzyme catalytically active by facilitating the removal of an inhibitory molecule bound to the aptamer of the third MNAzyme.

[0366] Example 38 The method of any one of embodiments 31 or 33 to 37, wherein:

[0367] - the reaction mixture comprises the third MNAzyme and either the first MNAzyme or the second MNAzyme; and

[0368] - the sequence of the loop portion of the third closed stem-loop oligonucleotide capable of hybridising to the substrate arm of the third MNAzyme is different from:

[0369] the sequence of the loop portion of the first closed stem-loop oligonucleotide capable of hybridizing to the substrate arm of the first MNAzyme, and / or

[0370] The sequence of the loop portion of the second closed stem-loop oligonucleotide is capable of hybridising to the substrate arm of the first MNAzyme.

[0371] Example 39 The method of any one of embodiment 28 or embodiments 31 to 38, wherein:

[0372] - the number of hybridizing nucleotides and / or the sequence of hybridizing nucleotides in the stem portion of the third closed stem-loop oligonucleotide is different from the number of hybridizing nucleotides and / or the sequence of hybridizing nucleotides in the stem portion of the first closed stem-loop oligonucleotide and the second closed stem-loop oligonucleotide;

[0373] - the third temperature is different from the first temperature and the second temperature; and

[0374] - the fluorophores of the third closed stem-loop oligonucleotide and the third open stem-loop oligonucleotide emit in the same color region of the visible spectrum as the fluorophores of the first closed stem-loop oligonucleotide and the second closed stem-loop oligonucleotide and the first open stem-loop oligonucleotide and the second open stem-loop oligonucleotide.

[0375] Example 40 . The method of any one of embodiment 28 or embodiments 31 to 39, wherein the fluorophore of the third closed and open stem-loop oligonucleotides is the same as the fluorophore of the first and / or second closed and open stem-loop oligonucleotides.

[0376] Example 41 .A method according to embodiment 40, wherein the fluorophore of the third closed and open stem-loop oligonucleotide and the fluorophore of the first closed and open stem-loop oligonucleotide and / or the fluorophore of the second closed and open stem-loop oligonucleotide can be detected using the same emission channel of the device.

[0377] Example 42 The method of any one of embodiments 28 to 38 or 39, wherein:

[0378] - the fluorophores of the first closed stem-loop oligonucleotide and the first open stem-loop oligonucleotide emit in the same color region of the visible spectrum as the fluorophores of the second closed stem-loop oligonucleotide and the second open stem-loop oligonucleotide; and

[0379] - the fluorophores of the third closed stem-loop oligonucleotide and the open stem-loop oligonucleotide emit in a different color region of the visible spectrum than the fluorophores of the first closed stem-loop oligonucleotide and the second closed stem-loop oligonucleotide and the first open stem-loop oligonucleotide and the second open stem-loop oligonucleotide.

[0380] Example 43.The method according to any one of embodiments 28 to 42, wherein the third temperature differs from the first temperature and / or the second temperature by more than: 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C.

[0381] Example 44 .A method according to any one of embodiments 28 to 43, wherein detection of a third fluorescent signal at the third temperature indicates the presence of the third target in the sample, and absence of detection of a third fluorescent signal at the third temperature indicates the absence of the third target in the sample.

[0382] Example 45 The method of any one of embodiments 1 to 44, wherein:

[0383] (i) determining that the presence of the first fluorescent signal at the first temperature indicates the presence of the first target in the sample, and determining that the absence of the first fluorescent signal indicates the absence of the first target in the sample; and

[0384] (ii) determining that the presence of the second fluorescent signal at the second temperature indicates the presence of the second target in the sample, and determining that the absence of the second fluorescent signal indicates the absence of the second target in the sample.

[0385] Example 46. Embodiment 44: The method of any one of embodiments 1 to 44, wherein determining the presence or absence of the first and second targets comprises using melting curve analysis of the first and second fluorescent signals.

[0386] Example 47 The method of any one of embodiments 1 to 44, wherein part (c) comprises detecting the levels of the first and second fluorescent signals under the following conditions:

[0387] - a temperature equal to or higher than said second temperature; and

[0388] - a temperature that is equal to or higher than the first temperature and lower than the second temperature.

[0389] Example 48 .A method according to embodiment 47, wherein part (c) further includes detecting the levels of the first and second fluorescent signals at a temperature lower than the second temperature.

[0390] Example 49.A method according to Example 47 or Example 48, wherein part (c) includes detecting the levels of the first and second fluorescent signals during and / or after the nucleic acid amplification reaction is completed.

[0391] Example 50 . The method of any one of embodiments 47 to 49, further comprising generating a first target positive control fluorescent signal using a known concentration of the first target and / or a known concentration of the first closed stem-loop oligonucleotide.

[0392] Example 51 . The method of any one of embodiments 47 to 50, further comprising generating a first target positive control fluorescent signal by repeating the method on a separate control sample comprising the first target.

[0393] Example 52 .A method according to embodiment 51, wherein the control sample comprising the first target comprises a known concentration of the first target.

[0394] Example 53 .The method according to Example 51 or Example 52, wherein the control sample comprising the first target further comprises the second target.

[0395] Example 54 . The method of any one of embodiments 46 to 53, further comprising generating a second target positive control fluorescent signal by repeating the method on a separate control sample comprising the second target.

[0396] Example 55 .A method according to embodiment 54, wherein the control sample comprising the second target includes a known concentration of the second target.

[0397] Example 56 .The method according to Example 54 or Example 55, wherein the control sample further includes the first target.

[0398] Example 57 . The method of any one of embodiments 46 to 53, further comprising generating a combined positive control fluorescent signal by repeating the method on a separate control sample comprising the first and second targets.

[0399] Example 58 .A method according to embodiment 57, wherein the combined control sample includes a known concentration of the first target and / or a known concentration of the second target.

[0400] Example 59.The method according to any one of embodiments 50 to 58, further comprising normalizing the first fluorescent signal and / or the second fluorescent signal using any of the positive control fluorescent signals.

[0401] Example 60 The method of any one of embodiments 46 to 59, further comprising generating a negative control fluorescent signal by repeating the method of any one of embodiments 1 to 3 on a separate negative control sample that does not contain:

[0402] (i) the first target; or

[0403] (ii) the second target; or

[0404] (iii) the first target or the second target.

[0405] Example 61 .The method according to Example 60 further includes normalizing the first fluorescent signal and / or the second fluorescent signal using the negative control fluorescent signal.

[0406] Example 62 The method of any one of embodiments 46 to 61, further comprising comparing the first and / or second fluorescent signal to a threshold value, wherein:

[0407] - generating the threshold using a fluorescent signal obtained from a series of samples tested according to the method described in Examples 1 to 3 and comprising any one or more of the following:

[0408] (i) No template control and the first target

[0409] (ii) No template control and the second target

[0410] (iii) no template control, the first target and the second target

[0411] To thereby determine the presence or absence of the first target and the second target in the sample.

[0412] Example 63 .A method according to embodiment 62, wherein the series of samples are tested using a known concentration of the first closed stem-loop oligonucleotide and / or a known concentration of the second closed stem-loop oligonucleotide.

[0413] Example 64.The method according to any one of embodiments 1 to 63, wherein the first temperature differs from the second temperature by more than: 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C.

[0414] Example 65 .A method according to one of embodiments 1 to 64, wherein any of the amplicons are produced by any one or more of the following: polymerase chain reaction (PCR), strand displacement amplification (SDA), helicase-dependent amplification (HDA), recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), rolling circle amplification (RCA), transcription-mediated amplification (TMA), self-sustained sequence replication (3SR), nucleic acid sequence-based amplification (NASBA) and / or reverse transcription polymerase chain reaction (RT-PCR).

[0415] Example 66 . The method according to one of embodiments 1 to 65, wherein the sample is a biological sample obtained from a subject.

[0416] Example 67 . The method according to one of embodiments 1 to 65, wherein the method is performed in vitro.

[0417] Example 68 . The method according to one of embodiments 1 to 65, wherein the method is performed ex vivo.

[0418] Example 69 A composition comprising:

[0419] a first closed stem-loop oligonucleotide and a second closed stem-loop oligonucleotide, wherein each of the closed stem-loop oligonucleotides comprises a double-stranded stem portion of hybridized nucleotides joined to a single-stranded loop portion of unhybridized nucleotides, wherein:

[0420] The first closed stem-loop oligonucleotide and the second closed stem-loop oligonucleotide differ in the number of hybridizing nucleotides and / or the sequence of hybridizing nucleotides in the stem portion, and

[0421] Each of the double-stranded stem portions comprises a fluorophore molecule attached to one strand and a quencher molecule attached to the opposite strand,

[0422] wherein the fluorophore molecules emit in the same color region of the visible spectrum and the melting temperature (Tm) of the double-stranded stem portion of the first closed stem-loop oligonucleotide is different from the Tm of the double-stranded stem portion of the second closed stem-loop oligonucleotide.

[0423] Example 70 . The composition of embodiment 69, wherein the single-stranded loop portion of the first closed stem-loop oligonucleotide is different in sequence from the single-stranded loop portion of the second closed stem-loop oligonucleotide.

[0424] Example 71 The composition of embodiment 69 or embodiment 70, further comprising:

[0425] a first MNAzyme comprising a substrate arm capable of hybridising to the closed single stranded loop portion of the first closed stem-loop oligonucleotide; and

[0426] A second MNAzyme comprising a substrate arm capable of hybridising to the single stranded loop portion of the second closed stem-loop oligonucleotide.

[0427] Example 72 The composition of embodiment 71, wherein:

[0428] the substrate arm of the first MNAzyme hybridizes to the single stranded loop portion of the first closed stem-loop oligonucleotide; and

[0429] The substrate arm of the second MNAzyme hybridizes to the single stranded loop portion of the second closed stem-loop oligonucleotide.

[0430] Example 73 The composition of embodiment 72, wherein:

[0431] the first MNAzyme and / or the second MNAzyme comprises an aptamer sequence that binds to a target analyte, protein, compound or molecule and a sensor arm that hybridizes to an oligonucleotide sequence; and

[0432] The first MNAzyme is designed to detect a different target than the second MNAzyme.

[0433] Example 74 The composition of embodiment 72, wherein:

[0434] The first MNAzyme and / or the second MNAzyme comprises a sensor arm that hybridizes to a target sequence, and the first MNAzyme is designed to detect a different target than the second MNAzyme.

[0435] Embodiment 75. The composition of any one of embodiments 69 to 74, further comprising:

[0436] a first oligonucleotide sequence that hybridizes to the single-stranded loop portion of the first closed stem-loop oligonucleotide that is different in sequence from the single-stranded loop portion of the second closed stem-loop oligonucleotide, thereby forming a double-stranded sequence including the first oligonucleotide;

[0437] a second oligonucleotide sequence that hybridizes to the single-stranded loop portion of the second closed stem-loop oligonucleotide, the second oligonucleotide sequence being different from the sequence of the single-stranded loop portion of the first closed stem-loop oligonucleotide, thereby forming a double-stranded sequence including the second oligonucleotide;

[0438] a restriction endonuclease that associates with and is capable of cleaving the double-stranded sequence comprising the first oligonucleotide; and / or

[0439] A restriction endonuclease that associates with and is capable of cleaving the double-stranded sequence comprising the second oligonucleotide.

[0440] Embodiment 76. The composition of any one of embodiments 69 to 75, further comprising:

[0441] a first double-stranded sequence comprising a first target oligonucleotide sequence that hybridizes to the single-stranded loop portion of the first closed stem-loop oligonucleotide having a sequence different from that of the single-stranded loop portion of the second closed stem-loop oligonucleotide, thereby forming a first double-stranded sequence comprising the first target oligonucleotide;

[0442] a second double-stranded sequence comprising a second target oligonucleotide sequence that hybridizes to the single-stranded loop portion of the second closed stem-loop oligonucleotide having a sequence different from that of the single-stranded loop portion of the first closed stem-loop oligonucleotide, thereby forming a double-stranded sequence comprising the second target oligonucleotide;

[0443] a first primer oligonucleotide that hybridizes upstream (5') to the first target oligonucleotide relative to the first double-stranded sequence, and a first enzyme having exonuclease activity (e.g., a polymerase, an exonuclease) that associates with the loop portion of the first closed stem-loop oligonucleotide at or near an end of the first primer oligonucleotide; and / or

[0444] a second primer oligonucleotide that hybridizes to the second target oligonucleotide upstream (5') relative to the second double-stranded sequence, and a second enzyme with exonuclease activity (e.g., a polymerase, an exonuclease) that associates with the loop portion of the second closed stem-loop oligonucleotide at or near an end of the second primer oligonucleotide.

[0445] Example 77 The composition of any one of embodiments 69 to 76, further comprising:

[0446] a first double-stranded sequence comprising a first target oligonucleotide sequence that hybridizes to the single-stranded loop portion of the closed first stem-loop oligonucleotide having a sequence different from that of the single-stranded loop portion of the second closed stem-loop oligonucleotide, thereby forming a first double-stranded sequence comprising the first target oligonucleotide;

[0447] a second double-stranded sequence comprising a second target oligonucleotide sequence that hybridizes to the single-stranded loop portion of the second closed stem-loop oligonucleotide having a sequence different from that of the single-stranded loop portion of the first closed stem-loop oligonucleotide, thereby forming a second double-stranded sequence comprising the second target oligonucleotide;

[0448] A first enzyme having exonuclease activity associates with the first double-stranded sequence, and a second enzyme having exonuclease activity associates with the second double-stranded sequence.

[0449] Example 78. The composition of any one of embodiments 69 to 77, further comprising:

[0450] a first DNA enzyme and / or a first ribozyme that hybridizes to a loop portion of the first closed stem-loop oligonucleotide that has a sequence different from that of the single-stranded loop portion of the second closed stem-loop oligonucleotide,

[0451] a second DNA enzyme and / or a second ribozyme that hybridizes to the loop portion of the second closed stem-loop oligonucleotide having a sequence different from that of the single-stranded loop portion of the first closed stem-loop oligonucleotide,

[0452] a cofactor for each of the DNA enzymes, the cofactor being capable of causing each of the DNA enzymes to exhibit catalytic activity and thereby inducing cleavage activity of any of the DNA enzymes hybridized to the loop portion.

[0453] a cofactor for each of said ribozymes, said cofactor being capable of rendering each of said ribozymes catalytically active and thereby inducing the cleavage activity of any of said ribozymes hybridized to said loop portion.

[0454] Example 79. The composition of any one of embodiments 69 to 78, further comprising:

[0455] a third closed stem-loop oligonucleotide, wherein the third closed stem-loop oligonucleotide comprises a double-stranded stem portion of hybridized nucleotides linked to a single-stranded loop portion of unhybridized nucleotides, wherein:

[0456] The number of hybridizing nucleotides and / or the sequence of hybridizing nucleotides in the stem portion of the third closed stem-loop oligonucleotide is different from the number of hybridizing nucleotides and / or the sequence of hybridizing nucleotides in the stem portion of the first closed stem-loop oligonucleotide and the second closed stem-loop oligonucleotide, and

[0457] The double-stranded stem portion of the third closed stem-loop oligonucleotide comprises a fluorophore molecule attached to one strand and a quencher molecule attached to the opposite strand, wherein the fluorophore molecule of the third closed stem-loop oligonucleotide emits in a different color region of the visible spectrum than the fluorophore molecules of the first closed stem-loop oligonucleotide and the second closed stem-loop oligonucleotide.

[0458] Example 80. The composition of any one of embodiments 69 to 78, further comprising:

[0459] a third closed stem-loop oligonucleotide, wherein the third closed stem-loop oligonucleotide comprises a double-stranded stem portion of hybridized nucleotides linked to a single-stranded loop portion of unhybridized nucleotides, wherein:

[0460] The number of hybridizing nucleotides and / or the sequence of hybridizing nucleotides in the stem portion of the third closed stem-loop oligonucleotide is different from the number of hybridizing nucleotides and / or the sequence of hybridizing nucleotides in the stem portion of the first closed stem-loop oligonucleotide and the second closed stem-loop oligonucleotide, and

[0461] The double-stranded stem portion of the third closed stem-loop oligonucleotide comprises a fluorophore molecule attached to one strand and a quencher molecule attached to the opposite strand, wherein the fluorophore molecule of the third closed stem-loop oligonucleotide emits in the same color region of the visible spectrum as the fluorophore molecules of the first closed stem-loop oligonucleotide and the second closed stem-loop oligonucleotide.

[0462] Example 81. The composition of any one of embodiments 69 to 78, further comprising:

[0463] a third closed stem-loop oligonucleotide, wherein the third closed stem-loop oligonucleotide comprises a double-stranded stem portion of hybridized nucleotides linked to a single-stranded loop portion of unhybridized nucleotides, wherein:

[0464] The number of hybridizing nucleotides and / or the sequence of hybridizing nucleotides in the stem portion of the third closed stem-loop oligonucleotide is the same as the number of hybridizing nucleotides and / or the sequence of hybridizing nucleotides in the stem portion of the first closed stem-loop oligonucleotide or the second closed stem-loop oligonucleotide, and

[0465] The double-stranded stem portion of the third closed stem-loop oligonucleotide comprises a fluorophore molecule attached to one strand and a quencher molecule attached to the opposite strand, wherein the fluorophore molecule of the third closed stem-loop oligonucleotide emits in a different color region of the visible spectrum than the fluorophore molecules of the first closed stem-loop oligonucleotide and the second closed stem-loop oligonucleotide. BRIEF DESCRIPTION OF THE DRAWINGS

[0466] Reference will now be made to the appendix listed below. Figure 1-8 Preferred embodiments of the present invention have been described by way of example only.

[0467] Figure 1 : Shown are the melting temperatures (Tm) of exemplary LOCS reporters and their intact closed locked form and cleaved or degraded open form. Figure 1 A, LHS) has a loop region, a stem region, and a fluorophore (F) quencher (Q) dye pair. Cleavage or degradation of the loop region can generate an open LOCS reporter structure ( Figure 1 A, RHS). The melting temperature (Tm) of the stem region of the intact LOCS is higher than the melting temperature (Tm) of the stem region in the open LOCS structure. Thus, the stem A of LOCS A will separate or melt at Tm 1. The Tm 1 is higher than Tm 2, which is the temperature at which the open LOCS stem A' melts ( Figure 1 B). Similarly, the Tm of stem B of intact LOCS B will melt at Tm 3. Said Tm 3 is higher than Tm 4, which is the temperature at which the open LOCS stem B' melts ( Figure 1 B) The presence of a negative peak at the temperature corresponding to the Tm of the open LOCS structure indicates the presence of a target that induces the opening of a specific LOCS. Since each stem within each specific open LOCS melts at a different temperature, multiple LOCS labeled with the same fluorophore can be analyzed simultaneously in a single reaction.

[0468] Figure 2An exemplary strategy for detecting targets using LOCS oligonucleotides is presented, which are universal and can be used to detect any target. In this scheme, the LOCS oligonucleotide contains a stem region, a fluorophore quencher dye pair, and a loop region. The loop region comprises a universal substrate for catalytic nucleic acids such as MNAzymes, also known in the art as PlexZymes. When the target sensor arms of the component partzymes are arranged adjacent to each other on the target, an MNAzyme is formed. The loop region of the LOCS oligonucleotide binds to the substrate binding arms of the MNAzyme, and the substrate within the loop is cleaved by the MNAzyme and a fluorescent signal is generated. The reaction can be cooled to allow the stem region of the opened LOCS to reanneal, and then the reaction can be subjected to a melting curve analysis to monitor fluorescence as the reaction is heated. The presence of a melting peak corresponding to the Tm of the open LOCS indicates the presence of the target for which the MNAzyme was assembled. The target can be detected directly, or target amplicons generated by a target amplification scheme can be detected.

[0469] Figure 3 An exemplary strategy for detecting a target using a LOCS oligonucleotide specific for the target is shown. The LOCS oligonucleotide contains a stem region, a fluorophore-quencher dye pair, and a loop region that includes a region complementary to the target amplicon. Figure 3 In the scheme shown in Figure A, the loop region of the LOCS oligonucleotide binds to the target amplicon during amplification. During primer extension, the exonuclease activity of the polymerase degrades the loop region, thereby generating a fluorescent signal in real time, but leaves the stem region intact. The stem regions are complementary to each other but not to their targets. After amplification, the reaction can be cooled to allow the stem of the degraded open LOCS structure to reanneal. A subsequent melting curve analysis can be performed to measure the temperature at which the stem region from the open LOCS melts and generates fluorescence. Figure 3 In the scheme shown in B, the loop region of the LOCS oligonucleotide includes 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, thereby keeping the target molecule intact. This opens the LOCS and generates a fluorescent signal. The reaction can then be cooled so that the stem of the cracked open LOCS structure can reanneal; and a melting curve analysis can then be performed to measure the temperature at which the stem region derived from the open LOCS melts. When combined with a target amplification method, the strategy can be used to directly detect the target sequence or can detect the target amplicon.

[0470] Figure 4 demonstrated the extraction of a target containing MgPa( Figure 4 A), or TV-Btub( Figure 4 C), or both MgPa and TV-Btub ( Figure 4E) PCR amplification obtained in reactions with 10,000 copies (black line), 40 copies (grey line), or 0 copies (dashed line) of the target MgPa ( Figure 4 B) TV-Btub( Figure 4 D) or both MgPa and TV-Btub ( Figure 4 F) shows melting curve profiles obtained after amplification from reactions containing 10,000 copies (black line) and 40 copies (grey line). The melting profile generated by opening LOCS-1 in the presence of the MgPa gene target contains peaks at melting temperatures of 35°C and 41°C; the melting profile generated by opening LOCS-2 in the presence of the TV-Btub gene target contains a peak corresponding to a melting temperature of 50°C; however, the melting profile generated by opening both LOCS-1 and LOCS-2 in the presence of both MgPa and TV-Btub gene targets contains three peaks at melting temperatures of 35°C, 41°C, and 50°C. The LOCS melting profile in the presence of MgPa (Tm = 35°C and 41°C) differs from the LOCS melting profile generated in the presence of the TV-Btub gene target (Tm = 50°C). Furthermore, the LOCS melting profile in the presence of both MgPa and TV-Btub gene targets (Tm = 35°C, 41°C, and 50°C) differs from the above-described melting profile in the presence of only a single gene target, indicating that both targets are detected.

[0471] Figure 5 (Above) shows the results of the experiment on the MgPa( Figure 5 A1)、HMPV( Figure 5 B1) and CT-ompA( Figure 5 Plots of PCR amplification obtained from reactions containing 10,000 copies (black line), 40 copies (grey line), or 0 copies (dashed line) of the C1) gene target. The results shown in the lower panel are from reactions containing MgPa( Figure 5 A2)、HMPV( Figure 5 B2) and CT-ompA( Figure 5 C2) Melting curve signatures obtained from reactions with 10,000 copies (black line) and 40 copies (grey line) of a gene target. These three targets were specifically detected using three different MNAzymes; each of which had the same substrate binding arm and differed only in the target binding arm. Each MNAzyme opened the same universal LOCS-1 oligonucleotide comprising a universal substrate and a universal stem. Each target produced a melting curve signature with a peak at 40°C, corresponding to the Tm of the universal stem.

[0472] Figure 6 (Above) shows the Figure 6 A1)、VZV( Figure 6B1) and rpoB( Figure 6 Plots of PCR amplification obtained from reactions containing 10,000 copies (black line), 40 copies (grey line), or 0 copies (dashed line) of the target C1). The results shown in the lower panel are from reactions containing TV-Btub ( Figure 6 A2)、VZV( Figure 6 B2) and rpoB( Figure 6 C2) Melting curve characteristics obtained from reactions with 10,000 copies (black line) and 40 copies (grey line) of a target. These three targets were specifically detected by three different MNAzymes; each of which had the same substrate binding arm and differed only in the target binding arm. Each MNAzyme opened the same universal LOCS-2 comprising a universal substrate and a universal stem. Each target produced a melting curve characteristic with a peak at 50°C, corresponding to the Tm of the universal stem within the LOCS-2.

[0473] Figure 7 Shown in the use of LOCS-1 ( Figure 7 A), LOCS-2 ( Figure 7 B) LOCS-3 Figure 7 C) and LOCS-4( Figure 7 D) Monitoring target CT-Cds ( Figure 7 A and 7C) or TFRC( Figure 7 Melting curve characteristics obtained upon amplification of LOCS-1, LOCS-2, LOCS-3, and LOCS-4 (Figures 7B and 7D). The melting curve characteristics obtained in the absence of target (dashed lines) have peaks at Tm of 65°C, 77°C, 66°C, and 67°C, corresponding to the melting temperatures of closed intact LOCS-1, LOCS-2, LOCS-3, and LOCS-4, respectively. In the absence of target (black lines; Figure 7 A and 7C), opening of the LOCS-1 and LOC-3 structures resulted in melting temperatures of 30°C and 36°C, respectively. Figure 7 B and 7D), opening of the LOCS-2 and LOC-4 structures resulted in melting temperatures of 50°C and 32°C, respectively.

[0474] Figure 8 Shown in CFX96 Thermal Cycler ( Figure 8 A) or ABI 7500( Figure 8 B) or Light Cycler480 (Figure C) or XXpress PCR ( Figure 8 Melting curves obtained when amplifying the MgPa target were performed on 400 nm CMOS instruments (D). Both targets were monitored in the FAM channel on all machines.

[0475] Figure 9 Shown in CFX96 Thermal Cycler ( Figure 9 A) or ABI 7500( Figure 9 B) or Lightcycler480( Figure 9 C) or XXpress PCR ( Figure 9 Melting curves obtained when amplifying the TV-Btub target were performed on 400 nm CMOS sensors (D). Both targets were monitored in the FAM channel on all machines.

[0476] Figure 10 Shown are melting curve characteristics obtained from a duplex PCR reaction in which the targets MgPa (black curve) and TV-Btub (grey curve) were co-amplified and read in a single channel. The solid and dashed lines indicate target concentrations of 10,000 copies and 40 copies, respectively. Figure 10 A shows the melting curves obtained using LOCS-1 and LOCS-2 read in the FAM channel; Figure 10 B shows the results using LOC-5 and LOCS-6 read in the HEX channel; Figure 10 C shows the results using LOCS-7 and LOCS-8 read in the Texas Red channel, and Figure 10 D shows the results using LOCS-9 and LOCS-10 read in the Cy5 channel.

[0477] Figure 11 shows the PCR amplification of the Figure 11 A) TV-Btub( Figure 11 B) CTcry Figure 11 C), MgPa and TV-Btub ( Figure 11 D), MgPa and CTcry ( Figure 11 E) or TV-Btub and CTcry( Figure 11 F) Melting curve profiles obtained from reactions of 10,000 copies of a gene target. Melting profiles generated by opening LOCS-1 and / or LOCS-2 and / or LOCS-11 produce peaks indicating that these structures melt in the range of 30°C to 50°C, while intact closed LOCS melt in the range of 64°C to 74°C. In each case, the LOCS melting profile is unique and distinct from the other LOCS melting profiles.

[0478] Figure 12Shown are the melting curve characteristics obtained from reactions containing 10,000 copies of each of the MgPa, TV-Btub, and CTcry gene targets after PCR amplification. The melting characteristics generated by opening LOCS-1, LOCS-2, and LOCS-11 in the presence of the MgPa, TV-Btub, and CTcry gene targets contain three peaks at melting temperatures of 30°C, 41°C, and 49°C. Reactions containing all three gene targets can be compared to reactions containing only a single gene target ( Figure 11 A-11C) and reactions containing two of the three gene targets ( Figure 11 For example, reactions containing all three gene targets can be distinguished from reactions containing only TV-Btub and CTcry by the disappearance of the Tm peak (approximately 64°C) of open LOCS 1. Figure 11 F) differentiate.

[0479] Figure 13 Melting curve signatures obtained from a quadruplex reaction containing 10,000 copies of either the MgPa, TV-Btub, NGopa, or gpd gene targets after PCR amplification are shown. These four targets were specifically detected using four different MNAzymes, which in turn cleaved and opened four different LOCS reporters, which were monitored in two fluorescence channels. Each LOCS reporter contained a different universal substrate, but the same universal stem (Stem 1 and Stem 2) was used in both the FAM and Texas Red channels. The presence of the MgPa or TV-Btub gene was detected by an increase in signal in the Texas Red channel; and the presence of the NGopa or gpd gene was detected by an increase in signal in the FAM channel. Specific detection of MgPa or TV-Btub in the Texas Red channel and NGopa or gpd in the FAM channel was determined based on unique melting curve signatures. The melting characteristics produced in the Texas Red channel by opening LOCS-7 in the presence of MgPa and opening LOCS-8 in the presence of TV-Btub contain peaks at melting temperatures of 43° C. and 53° C., respectively. The melting characteristics produced in the FAM channel by opening LOCS-12 in the presence of NGopa and opening LOCS-13 in the presence of gpd contain peaks at melting temperatures of 26° C., 42° C., and 53° C., respectively.

[0480] Figure 14 Shown are melting curve characteristics obtained from a singleplex PCR reaction where each of the loop regions of the three LOCS reporters contained a different substrate for a different MNAzyme, but all three contained the same stem sequence. The melting characteristic obtained in the absence of target (grey line) shows 74°C ( 10°C) corresponding to the melting temperatures of closed intact LOCS-2, LOCS-14, and LOCS-15, respectively. Figure 14 A), 75℃( Figure 14 B) and 75℃( Figure 14 C). In the TFRC target (black line; Figure 14 In the presence of 14A, 14B, and 14C), opening of the LOCS-2, LOCS-14, and LOC-15 structures resulted in melting temperatures of 49°C, 48°C, and 49°C, respectively.

[0481] Figure 15 The cleavage of LOCS-16 ( Figure 15 A) and LOCS-17( Figure 15 B) Melting curve characteristics obtained when used to detect and identify two different targets (AF-NE-TV1 and AF-NE-R5b). The melting curve characteristics obtained in the absence of target (grey line) have peaks at Tm of 65°C and 76°C, which correspond to the melting temperatures of closed intact LOCS-16 and LOCS-17, respectively. The melting curve characteristics obtained in the presence of target (black line, AF-NE-TV1 and AF-NE-R5b, respectively) have peaks at Tm of 29°C and 48°C, which correspond to the melting temperatures of cleaved open LOCS-16 and LOCS-17, respectively. The data from this example demonstrates that the LOCS reporter can be used with alternative target detection methods (such as methods using nicking endonucleases).

[0482] Figure 16 Shown are the amplification curves obtained when opening the LOCS probe using a strategy similar to that used to mediate TaqMan / hydrolysis signal generation ( Figure 16 A) and melting curve characteristics ( Figure 16 B), i.e., the target-specific region (loop) of the probe is degraded during PCR by the exonuclease activity of the polymerase. PCR profiles obtained from reactions containing 10,000 copies (black line) or 0 copies (grey line) of the TV-btub gene target ( Figure 16 A). The melting curve profile obtained in the absence of target (grey line) has a peak at Tm of 61 °C corresponding to the melting temperature of closed intact LOCS-18 ( Figure 16 B). The melting curve profile obtained in the presence of target (black line) has a peak at Tm of 40°C corresponding to the melting temperature of cleaved open LOCS-18 ( Figure 16 B) The data from this example demonstrates that the LOCS reporter can be used with alternative target detection methods such as the TaqMan method which uses an enzyme with exonuclease activity to cleave probes hybridized to the amplicon.

[0483] Figure 17 showed that at 39℃( Figure 17 A) and 72℃( Figure 17 B) PCR amplification plots obtained from reactions containing 20,000 copies of target X / CTcry (solid black line), 20,000 copies of target Y / NGopa (dashed black line), or 20,000 copies of both targets (gray line). Threshold values ​​TX and TY for the amplification plots obtained at 39° C. and 72° C., respectively, are indicated. Endpoint fluorescence values ​​designated EX1, EX2, EY1, and EY2 are indicated.

[0484] Figure 18 The results show that the expression of 20,000 copies of target Y / NGopa at 39°C ( Figure 18 A), 20,000 copies of target Y / NGopa at 72°C ( Figure 18 B), 20,000 copies of target Y / NGopa after normalization with FAF at 72°C ( Figure 18 C), 32 copies of target Y / NGopa at 39°C ( Figure 18 D), 32 copies of target Y / NGopa at 72°C ( Figure 18 E), 32 copies of target Y / NGopa after normalization with FAF at 72°C ( Figure 18 F), with 0 copies of target Y / NGopa at 39°C ( Figure 18 G), with 0 copies of target Y / NGopa at 72°C ( Figure 18 H), with 0 copies of target Y / NGopa after normalization with FAF at 72°C ( Figure 18 I) Plots of PCR amplification obtained in reactions containing 0 copies of target X / CTcry (solid black line), 32 copies of target X / CTcry (gray line), or 20,000 copies of target XCTcry (dashed black line).

[0485] Figure 19 Nonlinear logistic regression of the relative shift in Cq values ​​plotted against the copy number of In target Y (NGopa) - the copy number of In target X (CTcry) is shown.

[0486] Figure 20 Shown are the melting curve characteristics obtained in the HEX channel from a single-well 6-plex reaction containing 10,000 copies of either gpd, gpd3, or porA gene targets. Using a single well, six targets were specifically detected and differentiated using two fluorescence channels (three targets per channel). These targets were detected using six different MNAzymes, which in turn cleaved and opened six different LOCS reporters. Figure 20 A: LOCS-21 in the presence of gpd (53°C, 68°C and 85°C), Figure 20 B: LOCS-22 in the presence of gpd3 (30°C, 43°C, 77°C and 85°C), Figure 20 C: LOCS-23 in the presence of porA (68°C and 77°C), Figure 20 D: LOCS-21 and LOCS-22 in the presence of gpd and gpd3 (30°C, 43°C, 53°C and 85°C), Figure 20 E: LOCS-21 and LOCS-23 in the presence of gpd and porA (53°C and 68°C), Figure 20 F: LOCS-22 and LOCS-23 in the presence of gpd3 and porA (30°C, 43°C, 68°C and 77°C).

[0487] Figure 21 The results show that the Texas Red channel was generated from cells containing 10,000 copies of either TV-Btub, MgPa, or LGV gene targets. Figure 20 Figure 2. Melt curve characteristics obtained from the same single-well 6-plex reaction. In these reactions, six targets were specifically detected and differentiated using two fluorescence channels (three targets per channel). These targets were detected using six different MNAzymes, which in turn cleaved and opened six different LOCS reporters monitored in the HEX and Texas Red channels. Figure 21 A: Melting curve characteristics (30°C, 42°C, 66°C and 82°C) produced by LOCS-24 in the presence of TV-Btub, Figure 21 B: LOCS-25 in the presence of MgPa (53°C, 66°C and 82°C), Figure 21 C: LOCS-26 in the presence of LGV (68°C), Figure 21 D: LOCS-24 and LOCS-25 in the presence of TV-Btub and MgPa (30°C, 42°C, 53°C and 82°C), Figure 21 E: LOCS-24 and LOCS-26 in the presence of TV-Btub and LGV (30°C, 42°C and 68°C), Figure 21 F: LOCS-25 and LOCS-26 in the presence of MgPa and LGV (30°C, 43°C, 53°C and 68°C).

[0488] Figure 22 Shown are PCR amplifications from a gene containing 10,000 copies of each of gpd, gpd3, and porA ( Figure 22 A) or 10,000 copies of each of the TV-Btub, MgPa, and LGV gene targets ( Figure 22 B) Figure 20 and Figure 21Melting curve features obtained in the same 6-plex reaction. These targets were detected using six MNAzymes, which in turn cleave and open six LOCS reporters, which were monitored in two fluorescence channels. Figure 22 A: Melting profiles generated in HEX channels by LOCS-21, LOCS-22, and LOCS-23 in the presence of gpd, gpd3, and porA gene targets (30°C, 43°C, 53°C, and 68°C). Figure 22 B: Melting characteristics produced by LOCS-24, LOCS-25 and LOCS-26 in the presence of TV-Btub, MgPa and LGV in the Texas Red channel (30°C, 42°C, 53°C and 68°C).

[0489] Figure 23 shows the melting curve profiles obtained after PCR amplification from a 10-plex reaction containing 10,000 copies of any of the NGopa, porA, gpd, gpd3, TV-Btub, MgPa, CTcry, LGV, polA, or TFRC gene targets. The ten gene targets were specifically detected using ten different MNAzymes, which in turn cleaved and opened ten different LOCS reporters, which were monitored in five fluorescence channels (FAM, HEX, Texas Red, Cy5, and Cy5.5). Target differentiation within the FAM channel is shown in Figure 23A: LOCS-15 in the presence of NGopa (53°C), Figure 23B: LOCS-27 in the presence of porA (30°C and 76°C), and Figure 23C: LOCS-15 and LOCS-27 in the presence of NGopa and porA (30°C and 53°C). Discrimination of targets within the HEX channel is shown by FIG. 23D : LOCS-21 in the presence of gpd (53° C. and 70° C.), FIG. 23E : LOCS-22 in the presence of gpd3 (30° C., 43° C., and 76° C.), and FIG. 23F : LOCS-21 and LOCS-22 in the presence of gpd and gpd3 target genes (30° C., 43° C., and 53° C.). Discrimination of targets within the Texas Red channel is shown by FIG. 23G : LOCS-24 in the presence of TV-Btub (31° C., 41° C., 59° C., and 83° C.), FIG. 23H : LOCS-28 in the presence of MgPa (63° C.), and FIG. 23I : LOCS-24 and LOCS-28 in the presence of TV-Btub and MgPa (31° C., 41° C., and 63° C.). Discrimination of targets within the Cy5 channel is shown by: FIG23J: LOCS-29 in the presence of CTcry (61°C and 79°C), FIG23K: LOCS-10 in the presence of LGV (47°C and 80°C), and FIG23L: LOCS-29 and LOCS-10 in the presence of CTcry and LVG (47°C and 61°C). Discrimination of targets within the Cy5.5 channel is shown by: FIG23M: LOCS-30 and LOCS-31 in the presence of polA and TFRC gene targets (39°C and 59°C), and FIG23N: LOCS-31 in the presence of TFRC gene (39°C and 80°C).

[0490] Figure 24 Comparative analysis of the differential fluorescence levels between pre-amplification and post-amplification in the JOE channel from reactions containing 10,000 copies of TFRC, rpoB, pss, or all combinations of two or more gene targets, measured at 40.5°C, 56.5°C, and 78.5°C, is shown, expressed as the mean of the fluorescence levels of the triplicate reactions minus the mean of the triplicate reactions of the no-template control. Error bars are the standard deviation of each triplicate reaction.

[0491] Figure 25 Shown are comparative differential post-amplification fluorescence levels obtained in the JOE channel from reactions containing 10,000 copies of TFRC, rpoB, pss, or all combinations of two or more gene targets, measured at 32°C, 40.5°C, 56.5°C, and 65.5°C. The values ​​are expressed as the differential fluorescence levels between: (a) 32°C and 40.5°C; (b) 40.5°C and 56.5°C; and (c) 56.5°C and 65°C, averaged across triplicate reactions. Error bars are the standard deviation for each triplicate reaction.

[0492] Figure 26 A comparative analysis of differential pre-amplification and post-amplification fluorescence levels obtained in the JOE channel from reactions containing 10,000 copies of TFRC, rpoB, pss, or all combinations of two or more gene targets measured at 39.5°C, 56.5°C, and 65.5°C is shown. The values ​​are expressed as (i) the difference between pre-amplification and post-amplification fluorescence levels at 39.5°C (ii) the difference between pre-amplification and post-amplification fluorescence levels at 39.5°C and the difference between pre-amplification and post-amplification fluorescence levels at 56.5°C (iii) the difference between pre-amplification and post-amplification fluorescence levels at 56.5°C and the difference between pre-amplification and post-amplification fluorescence levels at 65.5°C, averaged over triplicate reactions. Error bars are standard deviations for each triplicate reaction.

[0493] Figure 27 Shown are comparative melting peak heights at 40°C, 51°C, and 65°C in the JOE lane obtained from reactions containing 10,000 copies of TFRC, rpoB, pss, or all combinations of two or more gene targets, averaged across triplicate reactions. Error bars are the standard deviation for each triplicate reaction.

[0494] definition

[0495] 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 "polynucleotide" also includes a plurality of polynucleotides.

[0496] As used herein, the term "comprising" means "including". Variants of the word "comprising", such as "comprise" and "comprises", have correspondingly varying meanings. Thus, for example, a polynucleotide that "comprises" a nucleotide sequence may consist solely of the nucleotide sequence or may include one or more additional nucleotides.

[0497] As used herein, the term "plurality" means more than one. In certain specific aspects or embodiments, a 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 and range derivable therein.

[0498] As used herein, the term "subject" includes any animal of economic, social or research importance, including cattle, horses, sheep, primates, birds and rodents. Thus, a "subject" can be a mammal, such as a human or a non-human mammal. Microbial subjects are also encompassed, including but not limited to bacteria, viruses, fungi / yeasts, protozoa and nematodes. "Subjects" according to the present invention also include infectious agents such as prions.

[0499] As used herein, the terms "polynucleotide" and "nucleic acid" are used interchangeably and refer to single-stranded or double-stranded polymers of deoxyribonucleotides 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, precursor and primary microRNA, other non-coding RNA, ribosomal RNA, derivatives thereof, amplicons thereof, or any combination thereof. By way of non-limiting example, the source of the nucleic acid can be selected from the group consisting of synthetic, mammalian, human, animal, plant, fungal, bacterial, viral, archaeal, or any combination thereof. Unless otherwise indicated, the terms "polynucleotide" and "nucleic acid" and "oligonucleotide" include reference to any specified sequence and the sequence complementary thereto.

[0500] 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" will be understood to include nucleic acid targets and non-nucleic acid targets, such as proteins, peptides, analytes, ligands, and ions (e.g., metal ions).

[0501] As used herein, the term "oligonucleotide" refers to a fragment of DNA or a nucleic acid molecule containing DNA, or RNA or a molecule containing RNA, or a combination thereof. Examples of oligonucleotides include nucleic acid targets; substrates, such as those that can be modified by an MNAzyme; primers, such as primers used for in vitro target amplification by methods such as PCR; and components of an MNAzyme. Unless otherwise indicated, the term "oligonucleotide" includes reference to any specified sequence as well as sequences complementary thereto. The oligonucleotide may include at least one addition or substitution including, but not limited to, the group consisting of: 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, 2'-O-methylcytidine, 5-carboxymethylaminomethylthiouridine, dihydrouridine, 2'-O-methylpseudouridine, βD-galactosylqueridine, 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, βD-mannosylmethyluridine, 5-methoxycarbonylmethyluridine, 5-methoxyuridine, 2-methylthio-N6-isopentenyladenosine, N- ((9-β-ribofuranosyl-2-methylthiopurin-6-yl)carbamoyl)threonine, N-((9-β-ribofuranosylpurin-6-yl)N-methyl-carbamoyl)threonine, uridine-5-hydroxyacetic acid methyl ester, uridine-5-hydroxyacetic acid (v), wybutoxosine, pseudouridine, queosine, 2-thiocytidine, 5-methyl-2-thiouridine, 2-thiouridine, 4-thiouridine, 5-methyluridine, N-((9-β-D-ribofuranosylpurin-6-yl)carbamoyl)threonine, 2'-O-methyl-5-methyluridine, 2'-O-methyluridine, wybutosine, 3-(3-amino-3-carboxypropyl)uridine, βD-arabinouridine, βD-arabinothymidine.

[0502] 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 by Watson-Crick base pairing or wobble base pairing. Bonds can be formed by Watson-Crick base pairing between adenine (A) bases and uracil (U) bases, between adenine (A) bases and thymine (T) bases, between cytosine (C) bases and guanine (G) bases. Wobble base pairs are non-Watson-Crick base pairings between two nucleotides in a polynucleotide duplex (e.g., guanine-uracil, inosine-uracil, inosine-adenine and inosine-cytosine). Nucleotides that are referred to as "complementary" or "complementary" to each other are nucleotides that have the ability to hybridize together by Watson-Crick base pairing or by wobble base pairing between their corresponding bases.

[0503] As used herein, the terms "non-complementary," "non-complementary," "mismatch," and "mismatched" refer to nucleotides (e.g., deoxyribonucleotides, ribonucleotides, and combinations thereof) that lack the ability to hybridize together by Watson-Crick base pairing or by wobble base pairing between their corresponding bases.

[0504] As used herein, "enzyme" refers to any molecule that can catalyze a chemical reaction (e.g., amplification of a polynucleotide, cleavage of a polynucleotide, etc.). Non-limiting examples of enzymes suitable for use in the present invention include nucleases and protein enzymes. Non-limiting examples of suitable nucleases include RNAzymes, MNAzymes, and DNA enzymes. Non-limiting examples of suitable protein enzymes include exonucleases and endonucleases. These enzymes typically provide catalytic activity that helps to implement one or more of the methods described herein. By way of non-limiting examples, exonuclease activity can be, for example, the inherent catalytic activity of a polymerase. By way of non-limiting examples, endonuclease activity can be, for example, the inherent catalytic activity of a restriction enzyme comprising a nicking endonuclease, an endoribonuclease, or a double-stranded specific nuclease (DSN).

[0505] 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, HDA, RPA, LAMP, RCA, TMA, 3SR, or NASBA.

[0506] As used herein, the term "stem-loop oligonucleotide" will be understood to mean a double-stranded stem component connected to a single-stranded loop component or a DNA or DNA-containing molecule, or RNA or RNA-containing molecule, or a combination thereof (i.e., a DNA-RNA hybrid molecule or complex). The double-stranded stem component includes a forward strand that hybridizes to a complementary reverse strand through complementary base pairing, wherein the 3' nucleotides of the forward strand are connected to the 5' nucleotides of the single-stranded loop component, and the 5' nucleotides of the reverse strand are connected to the 3' nucleotides of the single-stranded loop component. The double-stranded stem component may further include one or more fluorophores on one strand (e.g., the forward strand) and one or more quenchers on the opposite strand (e.g., the reverse strand).

[0507] As used herein, the terms "stem-loop oligonucleotide" and "LOCS" (also referred to herein as "LOCS oligonucleotide," "LOCS structure," "LOCS reporter," "complete LOCS," "closed LOCS," and "LOCS probe") are used interchangeably herein and will be understood to mean a double-stranded stem component (also referred to herein as "stem," "stem region," and "stem portion") connected to a single-stranded loop component (also referred to herein as "loop," "loop region," and "loop portion") comprising or consisting of 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). The double-stranded stem component comprises a forward strand hybridized to a complementary reverse strand by complementary base pairing, wherein the 3' nucleotide of the forward strand is connected to the 5' nucleotide of the single-stranded loop component, and the 5' nucleotide of the reverse strand is connected to the 3' nucleotide of the single-stranded loop component.

[0508] The double-stranded stem component may further comprise one or more fluorophores on one strand (e.g., the forward strand) and one or more quenchers on the opposite strand (e.g., the reverse strand). For example, one or more fluorophores may be attached at or near the 5' end of the forward strand and one or more quenchers may be attached at or near the 3' end of the reverse strand, or vice versa. The single-stranded loop component may comprise a region that is capable of serving as a substrate for a catalytic nucleic acid, such as an MNAzyme, a DNAzyme, a ribozyme, an aptamer MNAzyme, or an aptazyme. Additionally or alternatively, the single-stranded loop component may comprise 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 the region is further capable of serving as a substrate for a nucleic acid exonuclease. By way of non-limiting example, the exonuclease may be an intrinsic activity of a polymerase. Additionally or alternatively, the single-stranded loop component region may comprise a region that is (i) complementary to the target being detected; (ii) comprises one strand of a double-stranded restriction enzyme recognition site; and (iii) is capable of serving as a substrate for a restriction enzyme.

[0509] As used herein, the terms "open stem-loop oligonucleotide," "open LOCS," "open LOCS oligonucleotide," "open LOCS structure," "open LOCS reporter," "open LOCS probe," "open LOCS," "cleaved LOCS," and "degraded LOCS" are used interchangeably herein and will be understood to refer to a "stem-loop oligonucleotide" or "LOCS" in which the single-stranded loop component is cleaved and / or degraded (e.g., by an enzyme as described herein) such that at least one bond between adjacent nucleotides within the loop is removed, thereby providing an open structure in the loop region. In an open LOCS, the forward and reverse strands of the double-stranded stem portion can retain the ability to hybridize to each other to form a stem.

[0510] 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 LOCS containing loops that include a catalytic nucleic acid substrate or a sequence complementary to the target of interest. A single universal stem can be used as a surrogate marker for any target that can promote the opening of a specific LOCS. A series of universal stems can be incorporated into a series of LOCS designed to analyze any set of targets.

[0511] As used herein, the term "universal LOCS" refers to a LOCS structure containing a "universal stem" and a "universal loop" comprising 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 capable of facilitating the opening of a specific LOCS. A series of universal LOCS can be incorporated into any multiplex assay designed to analyze any set of targets.

[0512] 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 shall mean a DNA or DNA-containing molecule or complex, or an RNA or RNA-containing molecule or complex, or a combination thereof (i.e., 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 residue bases in the catalytic nucleic acid can comprise A, C, G, T, and U, as well as derivatives and analogs thereof. The above terms encompass unimolecular nucleases, which can include a single DNA or DNA-containing molecule (also referred to in the art as a "DNA enzyme," "deoxyribozyme," or "DNA enzyme") or an RNA or RNA-containing molecule (also referred to in the art as a "ribozyme") or a combination thereof (i.e., 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 encompass nucleases including DNA or DNA-containing complexes, or RNA or RNA-containing complexes, or combinations thereof (i.e., DNA-RNA hybrid complexes) that can recognize at least one substrate and catalyze modification (e.g., cleavage) of the at least one substrate. The terms "nuclease," "catalytic nucleic acid," "nucleic acid having catalytic activity," and "catalytic nuclease" encompass MNAzymes within their meaning.

[0513] As used herein, the terms "MNAzyme" and "multicomponent nucleic acid enzyme" are used herein to have the same meaning and refer to two or more oligonucleotide sequences (e.g., partzymes) that form an active nucleic acid enzyme capable of catalytically modifying a substrate only in the presence of an MNAzyme assembly facilitator (e.g., a target). "MNAzyme" is also known in the art as a "PlexZyme". MNAzymes can catalyze a range of reactions including cleavage of a substrate and other enzymatic modifications of one or more substrates. MNAzymes with endonuclease or cleavage activity are also referred to as "MNAzyme cleavage agents". The component partzymes, partzymes A and B, each bind to an assembly facilitator (e.g., a target DNA or RNA sequence) via Watson-Crick base pairing. An MNAzyme can only form when the sensor arms of partzymes A and B hybridize adjacent to each other on the assembly facilitator. The substrate arms of the MNAzyme engage the substrate and its modification (e.g., cleavage) is catalyzed by the catalytic core of the MNAzyme, which is formed by the interaction of the catalytic domains of partzymes A and B. MNAzymes can cleave DNA / RNA chimeric reporter substrates. MNAzyme cleavage of the substrate between a fluorophore and a quencher dye pair can generate a fluorescent signal. The terms "multicomponent nucleic acid enzyme" and "MNAzyme" encompass duplexes consisting of two molecules, triplicates consisting of three nucleic acid molecules, or other multiplexes, such as those consisting of four or more nucleic acid molecules.

[0514] It should be understood that the terms "MNAzyme" and "multicomponent nucleic acid enzyme" as used herein encompass all known MNAzymes and modified MNAzymes, including MNAzymes disclosed in any one or more of the following: 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 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 "multicomponent nucleic acid enzyme" include: MNAzymes with lytic 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 stabilizing oligonucleotides, MNAzymes comprising one or more activity inhibitors, multicomponent nucleic acid inactivating zymogens (MNAi), each of which is described in detail in one or more of the following: WO / 2007 / 041774, WO / 2008 / 040095, US 2007-0231810, US2010-0136536 and / or US 2011-0143338.

[0515] As used herein, the terms "partzyme", "component partzyme" and "partzyme component" refer to DNA-containing or RNA-containing or DNA-RNA-containing oligonucleotides, two or more of which can only together form an "MNAzyme" in the presence of an MNAzyme assembly facilitator as defined herein. In certain preferred embodiments, one or more component partzymes, and preferably at least two, may comprise three regions or domains: a "catalytic" domain, which forms part of the catalytically modified catalytic core; a "sensor arm" domain, which can associate and / or bind to the assembly facilitator; and a "substrate arm" domain, which can associate and / or bind to the substrate. The terms "sensor arm", "target sensor arm" or "target sensing arm" or "target arm" are 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 "aptamer partzyme". Partzymes may comprise a variety of components including, but not limited to, partzyme components having a truncated sensor arm and a stabilising arm component that stabilises the MNAzyme structure by interacting with an assembly facilitator or substrate.

[0516] As used herein, the terms "assembly facilitator molecule", "assembly facilitator", "MNAzyme assembly facilitator molecule" and "MNAzyme assembly facilitator" refer to an entity that facilitates the self-assembly of component partzymes to form catalytically active MNAzymes by interacting with the sensor arms of an MNAzyme. As used herein, an assembly facilitator may facilitate the assembly of an MNAzyme with cleavage or other enzymatic activity. In preferred embodiments, an assembly facilitator is required for the self-assembly of an MNAzyme. The assembly facilitator may consist of one molecule, or may consist of two or more "assembly facilitator components" that may pair or bind to the sensor arms of one or more oligonucleotide "partzymes". The assembly facilitator may include one or more nucleotide components that do not share sequence complementarity with one or more sensor arms of an MNAzyme. The assembly facilitator may be a target. The target can 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, precursor and primary microRNA, other non-coding RNA, ribosomal RNA, derivatives thereof, amplicons, or any combination thereof. The nucleic acid can be amplified. Amplification can include one or more of the following: PCR, RT-PCR, SDA, HDA, RPA, LAMP, RCA, TMA, 3SR, or NASBA.

[0517] As used herein, the term "detectable effect" is an effect that can be detected or quantified as an indication that cleavage of one or more LOCS probes has occurred. The magnitude of the effect can be indicative of the amount of input, such as an assembly facilitator (e.g., target). Detectable effects can be detected by a variety of methods, including fluorescence spectroscopy, surface plasmon resonance, mass spectrometry, NMR, electron spin resonance, polarized fluorescence spectroscopy, circular dichroism, immunoassays, chromatography, radioactivity measurement, photometry, scintigraphy, electronic methods, electrochemical methods, UV, visible or infrared spectroscopy, enzymatic methods, or any combination thereof.

[0518] As used herein, the terms "polynucleotide substrate" and "substrate" include any single-stranded or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases that can be recognized, acted upon or modified by an enzyme comprising a catalytic nucleic acid enzyme, or an analog, derivative, variant, fragment or combination thereof. A "polynucleotide substrate" or "substrate" can be modified by various enzymatic activities, including but not limited to cleavage. Cleavage or degradation of a "polynucleotide substrate" or "substrate" can provide a "detectable effect" for monitoring the catalytic activity of an enzyme. A "polynucleotide substrate" or "substrate" can be cleaved or degraded by one or more enzymes, including but not limited to catalytic nucleic acid enzymes such as MNAzymes, AptaMNAzymes, DNA enzymes, Aptazymes, ribozymes and / or protein enzymes such as exonucleases or endonucleases.

[0519] As used herein, a "reporter substrate" is a substrate that is particularly suitable for facilitating the measurement of the appearance of substrate cleavage or cleavage products associated with a catalytic reaction. The reporter substrate can be free in solution or, for example, bound (or "bound") to a surface or another molecule. The reporter substrate can 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 radioactive label, biotin (e.g., biotinylation), or a chemiluminescent label.

[0520] As used herein, a "universal substrate" is a substrate (e.g., a reporter substrate) that can be recognized or catalytically acted upon by a variety of MNAzymes, each of which can recognize a different assembly facilitator. The use of such substrates facilitates the development of separate assays for the detection, identification, or quantification of a variety of assembly facilitators using structurally related MNAzymes that all recognize the universal substrate. These universal substrates can each be individually labeled with one or more labels. In a preferred embodiment, one or more universal substrates are labeled with individually detectable labels to allow for the creation of a convenient system for detecting a variety of assembly facilitators independently or simultaneously using MNAzymes. In some embodiments, the substrate is capable of being catalytically modified by a DNA enzyme that is catalytically active in the presence of a cofactor (e.g., a metal ion cofactor such as lead or mercury).

[0521] The term "probe" as used herein refers to an oligonucleotide used to detect a target nucleic acid. Non-limiting examples of probes include TaqMan probes; molecular beacon probes; and LOCS probes, which include a nuclease substrate within a loop region that can be cleaved by a nuclease.

[0522] The term "product" refers to one or more new molecules produced as a result of enzymatic modification of a substrate. As used herein, the term "cleavage product" refers to a new molecule produced as a result of enzymatic cleavage or endonuclease activity. In some embodiments, the product of enzymatic cleavage or degradation of an intact closed LOCS structure comprises two oligonucleotide fragments capable of hybridizing to form an open LOCS structure.

[0523] As used herein, the use of the terms "melting temperature" and "Tm" in the context of polynucleotides will be understood to refer to the melting temperature (Tm) calculated using Wallace's rule, where Tm = 2°C (A+T) + 4°C (G+C) (see Wallace et al., (1979) Nucleic Acids Res. 6, 3543), unless specifically stated otherwise. The effect of sequence composition on melting temperature can be understood using the nearest neighbor method, which depends on 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 ion concentrations increase the chance of duplex formation, which results in an increase in melting temperature. Conversely, lower oligonucleotide and / or ion concentrations favor the dissociation of the stem, which results in a decrease in melting temperature.

[0524] As used herein, the term "quencher" includes any molecule that, when in close proximity to a fluorophore, absorbs the emission energy generated by the fluorophore and dissipates the energy as heat or emits light of 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 blackberry quenchers (BBQ).

[0525] As used herein, the term "base" when used in the context of nucleic acids will be understood to have the same meaning as the term "nucleotide."

[0526] As used herein, the term "test kit" refers to any delivery system for delivering materials. Such delivery systems include systems that allow reaction reagents (e.g., labels in appropriate containers, reference samples, support materials, etc.) and / or support materials (e.g., buffers, written instructions for performing assays, etc.) to be stored, transported, or delivered from one location to another. For example, a test kit can include one or more housings (e.g., boxes) containing relevant reaction reagents and / or support materials. The term "test kit" includes fragmentation test kits and combined test kits.

[0527] As used herein, the term "fragmentation kit" refers to a delivery system comprising two or more separate containers, each containing a subset of the complete kit components. The containers can be delivered to the intended recipient together or individually. Any delivery system comprising two or more separate containers, each containing a subset of the complete kit components, is included within the meaning of the term "fragmentation kit."

[0528] As used herein, a "combined kit" refers to a delivery system that contains all of the components of an assay in a single container (eg, in a single box containing each of the desired components).

[0529] It will be understood that use of the term "about" herein with reference to a recited value includes the recited value and values ​​within plus or minus ten percent of the recited value.

[0530] It will be understood that when referring to a numerical range, the term "between" is used herein to encompass the values ​​at each endpoint of the range. For example, a polypeptide having a length of between 10 residues and 20 residues includes a polypeptide having a length of 10 residues and a polypeptide having a length of 20 residues.

[0531] Any description herein of prior art documents or statements herein derived from or based upon these documents is not an admission that the documents or derived statements are part of the common general knowledge in the relevant art.

[0532] For the purposes of description, all documents referenced herein are hereby incorporated by reference in their entirety unless otherwise stated.

[0533] abbreviation

[0534] The following abbreviations are used herein and throughout the specification:

[0535] LOCS: Loops connected to the stem

[0536] MNAzyme: Multicomponent or concatemeric nuclease;

[0537] Partzymes: Partzymes containing oligonucleotides;

[0538] PCR: polymerase chain reaction;

[0539] gDNA: genomic DNA

[0540] NTC: No template control

[0541] qPCR: Real-time quantitative PCR

[0542] Ct; threshold cycle

[0543] R 2 ; Correlation coefficient

[0544] nM; nanomolar

[0545] mM; millimole

[0546] μL; microliter

[0547] dNTP; deoxynucleoside triphosphate

[0548] NF-H2O: nuclease-free water;

[0549] LNA: locked nucleic acid;

[0550] F: fluorophore;

[0551] Q: quencher;

[0552] N = A, C, T, G or any analog thereof;

[0553] N' = any nucleotide that is complementary to or capable of base pairing with N;

[0554] (N) x : any number of N;

[0555] (N') x : any number of N';

[0556] W: A or T;

[0557] R: A, G or AA;

[0558] rN: any ribonucleotide base;

[0559] (rN) x : any number of rN;

[0560] rR: A or G;

[0561] rY: C or U;

[0562] M: A or C;

[0563] H: A, C or T;

[0564] D: G, A or T;

[0565] JOE or 6-JOE: 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein;

[0566] FAM or 6-FAM: 6-carboxyfluorescein.

[0567] BHQ1: Black Hole Quencher 1

[0568] BHQ2: Black Hole Quencher 2

[0569] RT-PCR: reverse transcription polymerase chain reaction

[0570] SDA: Strand Displacement Amplification

[0571] HDA: helicase-dependent amplification

[0572] RPA: Recombinase polymerase amplification

[0573] LAMP: loop-mediated isothermal amplification

[0574] RCA: rolling circle amplification

[0575] TMA: transcription-mediated amplification

[0576] 3SR: self-sustaining sequence replication

[0577] NASBA: Nucleic Acid Sequence-Based Amplification

[0578] IB: Iowa FQ

[0579] IBR: Iowa RQ

[0580] shRNA: short hairpin RNA

[0581] siRNA: short interfering RNA

[0582] mRNA: messenger RNA

[0583] tRNA: transfer RNA

[0584] snoRNA: small nucleolar RNA

[0585] stRNA: small sequence RNA

[0586] smRNA: small regulatory RNA

[0587] pre-microRNA: precursor microRNA

[0588] pri-microRNA: primary microRNA

[0589] LHS: Left Hand Side

[0590] RHS: Right Hand Side

[0591] DSO: double-stranded oligonucleotide

[0592] Tm: melting temperature

[0593] RFU: relative fluorescence units DETAILED DESCRIPTION

[0594] The following detailed description conveys exemplary embodiments of the present invention in sufficient detail to enable one of ordinary skill in the art to practice the invention. The features or limitations of the various embodiments described do not necessarily limit other embodiments of the invention or the invention as a whole. Therefore, the following detailed description does not limit the scope of the invention, which is defined solely by the claims.

[0595] The present invention relates to methods and compositions for improved multiplexed detection of targets, such as nucleic acids, proteins, analytes, compounds, molecules, etc. The methods and compositions each employ LOCS oligonucleotides, which can be used in combination with various other reagents.

[0596] -LOCS oligonucleotides

[0597] Figure 1 Exemplary LOCS oligonucleotides of the invention are shown. Exemplary complete (closed) LOCS oligonucleotides ( Figure 1 A, LHS) has a loop region, a stem region, and a fluorophore (F) / quencher (Q) dye pair. Figure 1 Two exemplary LOCS oligonucleotides are shown, representing a complete LOCS A and a complete LOCS B. These LOCS oligonucleotides have different loop sequences and different stem sequences. When used in combination, two given LOCS oligonucleotides of the present invention typically differ in the sequence and / or length of the stem. By way of non-limiting example only, the stem A of the complete LOCS A can be designed to melt at a first temperature, representing Tm 1; while the stem B of the complete LOCS B can be designed to melt at a different temperature, representing Tm 3. Cleavage or degradation of the loop region of the complete LOCS oligonucleotide results in an open LOCS structure ( Figure 1 A, RHS).

[0598] Since intramolecular bonds are stronger than intermolecular bonds, the stem region of an intact LOCS structure will generally melt at a higher temperature than the stem of an open, cleaved or degraded LOCS oligonucleotide structure. For example, stem A of an intact LOCS A will melt at Tm 1, which is higher than Tm 2, the temperature at which the open LOCS stem A' melts ( Figure 1 B). Similarly, the melting temperature of stem B of the intact LOCS B will melt at Tm 3, which is higher than Tm 4, which is the temperature at which the open LOCS stem B' melts ( Figure 1 B). At the temperature corresponding to a specific open LOCS structure, the presence of a peak (e.g., detected by an associated fluorescent marker) demonstrates the presence of a target or target amplicon, which directs the opening of this specific LOCS structure. Since the stem of open LOCS A melts at a different temperature than open LOCS B ( Figure 1B), thus, multiple open LOCS structures labeled with the same fluorophore can be detected and analyzed simultaneously in a single reaction. Furthermore, multiple open LOCS structures labeled with different fluorophores can also be detected and analyzed simultaneously in a single reaction.

[0599] Now refer to Figure 2 In the exemplary embodiment depicted in , the sequence of the loop region of the LOCS oligonucleotide may be a substrate for, for example, an MNAzyme or one or more other catalytic nucleic acids. Figure 3 A and Figure 3 In a further embodiment shown in Figure B, the loop region of the LOCS oligonucleotide can be a target-specific sequence that is fully or partially complementary to the target to be detected and, when double-stranded, can serve as a substrate for degradation by an exonuclease (e.g., by the intrinsic exonuclease activity of a polymerase). Figure 3 In yet another exemplary embodiment, shown in Figure B, the target-specific sequence within the loop can further include a strand of a double-stranded restriction enzyme recognition site. Hybridization of the loop sequence to the target sequence can generate a functional, cleavable restriction site. In a preferred embodiment, the restriction enzyme is a nicking enzyme that is capable of cleaving the loop strand of the LOCS oligonucleotide while leaving the target intact.

[0600] In certain embodiments, the LOCS oligonucleotides of the present invention can be used to directly detect a target. In other exemplary embodiments, the LOCS can be used to detect target amplicons generated by target amplification techniques including, but not limited to, PCR, RT-PCR, SDA, HDA, RPA, LAMP, RCA, TMA, 3SR, or NASBA. The cleavage or degradation that results in the opening of the LOCS can occur in real time during target amplification or can be performed at the end point of the reaction after amplification. The loop region can be opened by target-dependent cleavage or degradation mediated by the enzymatic activity of a catalytic nucleic acid (including, but not limited to, an MNAzyme, a DNA enzyme, a ribozyme) or a protease including an exonuclease or endonuclease. By way of non-limiting example, the exonuclease activity can be, for example, the inherent catalytic activity of a polymerase. By way of non-limiting example, the endonuclease activity can be, for example, the inherent catalytic activity of a restriction enzyme including, for example, a nicking endonuclease, an endoribonuclease, or a double-strand specific nuclease (DSN).

[0601] Figure 2An exemplary strategy is presented in which the loop region comprises a substrate for a catalytic nucleic acid. In this strategy, 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, and an intervening loop region comprising a universal substrate for a catalytic nucleic acid such as an MNAzyme. The MNAzyme can detect the target directly or can be used to detect amplicons produced during target amplification. An MNAzyme is formed when the target sensor arms of the partzymes are arranged adjacent to each other on a target or target amplicon to form an active catalytic core. The loop region of the LOCS oligonucleotide binds to the substrate binding arms of the MNAzyme, and the substrate within the loop is cleaved by the MNAzyme, thereby opening the LOCS and generating a fluorescent signal. The reaction can then be cooled to allow the stem of the open LOCS structure to reanneal. The reaction can then be heated and a melting curve analysis performed to measure the temperature at which the open LOCS stem region melts. Those skilled in the art will recognize that the target can be detected in real time or at the end of the reaction.

[0602] Reactions designed to simultaneously detect multiple targets can contain multiple LOCS oligonucleotides; each of the multiple LOCS oligonucleotides includes a different universal substrate within the loop and a different stem region capable of melting at different temperatures following cleavage of the substrate / loop by a different MNAzyme. The LOCS oligonucleotides can further include the same fluorophore / quencher dye pair. By way of example, MNAzyme 1 can be formed in the presence of target 1 and cleave substrate 1 within LOCS oligonucleotide 1. This produces a cleaved double-stranded open LOCS structure 1 containing stem 1 that melts at temperature 1. Simultaneously, MNAzyme 2 can be formed in the presence of target 2 and cleave substrate 2 within LOCS oligonucleotide 2, thereby producing a cleaved double-stranded open LOCS structure 2 containing stem 2 that melts at temperature 2. When analyzing the melting curves of the reactions, the presence of a peak at temperature 1 indicates the presence of target 1; the presence of a peak at temperature 2 indicates the presence of target 2; and the presence of two peaks at temperatures 1 and 2 indicates the presence of both targets 1 and 2. In this way, a single wavelength read in a single channel of the instrument can detect and discriminate between the two targets.

[0603] The reaction mixture may further contain additional LOCS labeled with different fluorophore and quencher pairs. By way of example, LOCS oligonucleotides 1 and 2 may be labeled with fluorophore A, and LOCS oligonucleotides 3 and 4 may be labeled with fluorophore B. MNAzyme 1 may be formed in the presence of target 1 and cleave substrate 1 within LOCS oligonucleotide 1, thereby generating a cleaved double-stranded open LOCS structure 1 containing stem 1 that melts at temperature 1. MNAzyme 2 may be formed in the presence of target 2 and cleave substrate 2 within LOCS oligonucleotide 2, thereby generating a cleaved double-stranded open LOCS structure 2 containing stem 2 that melts at temperature 2. MNAzyme 3 may be formed in the presence of target 3 and cleave substrate 3 within LOCS oligonucleotide 3, thereby generating a cleaved double-stranded open LOCS structure 3 containing stem 3 that melts at temperature 3. MNAzyme 4 may be formed in the presence of target 4 and cleave substrate 4 within LOCS oligonucleotide 4, thereby generating a cleaved double-stranded open LOCS structure 4 containing stem 4 that melts at temperature 4. When the melting curve of the reaction is analyzed at the excitation wavelength of fluorophore A, the presence of a peak at temperature 1 indicates the presence of target 1; the presence of a peak at temperature 2 indicates the presence of target 2; and the presence of two peaks at temperatures 1 and 2 indicates the presence of both target 1 and target 2. When the melting curve of the reaction is analyzed at the excitation wavelength of fluorophore B, the presence of a peak at temperature 3 indicates the presence of target 3; the presence of a peak at temperature 4 indicates the presence of target 4; and the presence of two peaks at temperatures 3 and 4 indicates the presence of both target 3 and target 4. Thus, analysis at two wavelengths read in two channels of the instrument is able to detect and discriminate four targets. The skilled artisan will recognize that the described strategy can be expanded to monitor the cleavage of more than two targets at a particular wavelength, and that the number of fluorophores analyzed can be further increased to a number determined by the maximum capability of the instrument available to discriminate at each wavelength.

[0604] Now refer to Figure 3 The exemplary embodiments depicted illustrate two exemplary strategies for detecting a target using LOCS oligonucleotides having loop regions that are specific and complementary to the target and / or the amplicon. Figure 3 In the embodiment shown in Figure A, a LOCS oligonucleotide contains a stem region, a fluorophore-quencher dye pair, and a loop region that includes a region complementary to the target amplicon. During amplification, the loop region of the LOCS oligonucleotide binds to the target amplicon. During primer extension, the exonuclease activity of the polymerase degrades the loop region, leaving the stem region intact and generating a fluorescent signal in real time. After amplification, the reaction can be cooled to allow the stem of the degraded open LOCS structure to reanneal; and a melting curve analysis can then be performed to measure the temperature at which the stem region derived from the degraded open LOCS melts. Those skilled in the art will recognize that the target can be detected in real time or at the end of the reaction.

[0605] exist Figure 3 In another exemplary embodiment described in B, the LOCS oligonucleotide contains a stem region, a fluorophore / quencher dye pair and a loop region, wherein the loop region includes a region complementary to the target and further includes a recognition site for a restriction enzyme (e.g., a nicking enzyme). When the loop region of the LOCS oligonucleotide binds to the target, the nicking enzyme cleaves the loop chain of the LOCS, thereby keeping the target intact and generating a fluorescent signal. The reaction can be cooled so that the complete stem of the cracked open LOCS structure can be reannealed; and then a melting curve analysis can be performed to measure the temperature at which the stem region of the cracked open LOCS melts. Those skilled in the art will readily recognize that the target can be detected directly in the reaction without prior amplification. Further, those skilled in the art will recognize that the target amplicon produced by target amplification can be detected in real time or at the end of the reaction. In a preferred embodiment, the restriction enzyme used in conjunction with target amplification can be active and thermostable at one or more reaction temperatures.

[0606] In another exemplary embodiment, a LOCS oligonucleotide may contain a stem region, a fluorophore / quencher dye pair, and a loop region that may include a substrate for a DNA enzyme or ribozyme (e.g., a DNA enzyme or ribozyme that is catalytically active only in the presence of a metal ion). It is known in the art that certain DNA enzymes and ribozymes require metal cation cofactors to achieve catalytic activity. For example, some DNA enzymes and ribozymes are catalytically active only in the presence of, for example, lead or mercury. Such metals may be present, for example, in environmental samples. If a LOCS oligonucleotide contains a loop that includes a substrate for a DNA enzyme or ribozyme (e.g., one that is lead-dependent), the presence of lead in the sample may cause the LOCS to cleave and generate a fluorescent signal. The reaction may be cooled so that the intact stem of the cleaved, open LOCS structure can reanneal; and a melting curve analysis may then be performed to measure the temperature at which the stem region of the cleaved, open LOCS melts. One skilled in the art will readily recognize that multiple DNA enzymes and / or ribozymes, each dependent on a specific metal cofactor, may be combined in a single reaction designed to detect multiple targets (e.g., lead and mercury). Furthermore, one skilled in the art will recognize that the targets may be detected in real time or at the end of the reaction.

[0607] Reference to a nucleotide sequence being "substantially complementary" to another nucleotide sequence herein 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.

[0608] A nucleotide sequence that is "complementary" to another nucleotide sequence herein can mean that the first sequence is 100% 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.

[0609] Reference to a nucleotide sequence that is "not substantially complementary" to another nucleotide sequence herein can mean that the first sequence is less than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 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.

[0610] A nucleotide sequence that is "not complementary" to another nucleotide sequence herein 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.

[0611] Non-limiting examples of target nucleic acids (i.e., polynucleotides) that can be detected using LOCS oligonucleotides can include DNA, methylated DNA, alkylated DNA, complementary DNA (cDNA), RNA, methylated RNA, microRNA, siRNA, shRNA, mRNA, tRNA, snoRNA, stRNA, smRNA, precursor and primary microRNA, other non-coding RNA, ribosomal RNA, derivatives thereof, amplicons thereof, or any combination thereof (comprising mixed polymers of deoxyribonucleotide and ribonucleotide bases).

[0612] In some embodiments, the melting temperature ("Tm") of the intact LOCS oligonucleotide is higher than the Tm of the open LOCS structure.

[0613] Fluorescence signal analysis

[0614] According to the methods of the present invention, the fluorescent signal generated by the dissociation of the open LOCS structure can be analyzed in any suitable manner to detect, distinguish and / or quantify the target molecule.

[0615] While standard melting curve analysis can be used, various other methods of analysis that can be readily adapted for use in various assay formats are disclosed and exemplified herein (see Examples).

[0616] By way of non-limiting example, measurements of the fluorescence signal at a single temperature or multiple temperatures can be obtained at various time points within a reaction suitable for detecting cleavage or degradation of the loop region of the LOCS oligonucleotide. By way of non-limiting example, these time points can include (i) a time point at the start of the reaction, and / or (ii) a single time point or multiple time points during the course of the reaction; and / or (iii) a time point at the end or termination of the reaction.

[0617] In some embodiments, measurements of the fluorescence signal can be obtained at two or more temperatures for each cycle during PCR amplification. Analysis can be performed by comparing the fluorescence levels obtained at the first and / or second temperature and / or at another temperature.

[0618] In other embodiments, fluorescence signal measurements can be obtained at two or more temperatures for 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 specific temperature, and the Cq value can be measured as the cycle number at which the amplification plot exceeds the threshold. In embodiments where fluorescence signal measurements are obtained at two temperatures for each cycle during PCR, the Cq measured using the fluorescence signal at the lower temperature can allow for direct quantification of the starting concentration of the first target; and, as illustrated in Example 12, the Cq measured using the fluorescence signal at the higher temperature can be analyzed, thereby allowing for quantification of the starting concentration of the second target.

[0619] By way of non-limiting example, post-amplification measurement of fluorescence at two temperatures relative to a no-template control allows for specific detection of first and second cleaved open LOCS, the two temperatures comprising: a first temperature equal to or greater than the melting temperature of the first open LOCS and lower than the melting temperature of the second open LOCS; and a second temperature equal to or greater than the melting temperature of the second open LOCS. As demonstrated in Example 15 (Analytical Method A), at the first temperature, cleavage of the first LOCS produces a significant fluorescent signal relative to the no-template control reaction that exceeds a predetermined threshold. Due to the higher melting temperatures of the closed first and second LOCS and the open second LOCS, at this first temperature, the intact closed first and second LOCS and / or the cleaved open second LOCS do not contribute to a significant fluorescent signal that does not exceed the predetermined threshold. At a second temperature, higher than the first temperature, cleavage of the second LOCS produces a significant fluorescent signal that does not exceed the predetermined threshold relative to the no-template control, while the intact closed first and second LOCS and / or the open first LOCS do not contribute to a significant fluorescent signal that does not exceed the predetermined threshold relative to the no-template control. Detection of each cleaved LOCS reporter indicates the presence of the corresponding target in the sample.

[0620] By way of non-limiting example, post-amplification measurements of fluorescence relative to a control baseline fluorescence at specific temperature points allow for specific detection of the first and second cleaved open LOCS, wherein the specific temperature points include: a first temperature that is equal to or greater than the melting temperature of the first closed LOCS and less than the melting temperature of the second closed LOCS; and a second temperature that is equal to or greater than the melting temperature of the second closed LOCS.

[0621] As demonstrated in Example 15 (Analysis Method B), a control baseline fluorescence can be obtained by measuring fluorescence at a temperature or lower temperature at which neither open nor closed LOCS produces a significant fluorescence signal (temperature-0). Analysis can be performed by comparing the fluorescence levels obtained at the first and / or second temperature and temperature-0 and comparing these relative fluorescence levels with a predetermined threshold value. As demonstrated in Example 15, at a first temperature, the cracking of the first LOCS produces a significant fluorescence signal relative to the signal at temperature 0 and exceeds a predetermined threshold value. At this first temperature, the first and second LOCS that are completely closed and / or the open second LOCS that are cracked do not contribute to the generation of a significant fluorescence signal and do not exceed a predetermined threshold value. This is due to the higher melting temperatures of the closed first and second LOCS and the open second LOCS. At a second temperature that is higher than the first temperature, the cracking of the second LOCS produces a significant fluorescence signal relative to the signal obtained at temperature 0 and / or relative to the first temperature, and the signal shown exceeds a predetermined threshold value. At this second temperature, the completely closed first and / or second LOCS and the open first LOCS do not contribute to a significant fluorescent signal relative to the signal at temperature 0 and / or the first temperature and do not exceed a predetermined threshold. Detection of each cleaved LOCS reporter indicates the presence of the corresponding target in the sample.

[0622] By way of non-limiting example, a control baseline fluorescence can also be obtained by measuring fluorescence at the first and second temperatures at another time point at the start of the reaction (e.g., before PCR). At this other time point, all LOCS are intact (closed) and do not produce a significant fluorescence signal and do not exceed a predetermined threshold. Analysis can be performed by comparing the fluorescence levels obtained at the first and second temperatures at the time point when the reaction begins with the fluorescence levels obtained at the first and second temperatures during and / or after the reaction (e.g., during or after PCR). As shown in Example 15 (Analysis Method C), at the time point after the first temperature and PCR, the cleavage of the first LOCS produces a significant fluorescence signal relative to the signal of the intact, closed first LOCS measured at the time point before the first temperature and PCR. This relative signal exceeds a predetermined threshold. Due to the higher melting temperatures of the closed first and second LOCS and the open second LOCS, at this first temperature, the intact, closed first and second LOCS and / or the cleaved, open second LOCS do not contribute to the generation of a significant fluorescence signal relative to the signal obtained before PCR. At a second temperature higher than the first temperature and at the time point after PCR, the cleavage of the second LOCS produces a significant fluorescence signal relative to the signal obtained at the second temperature before PCR. At this second temperature and at a time point after PCR, the intact closed first or second LOCS and / or open first LOCS will not contribute to a significant fluorescent signal relative to the time point before PCR at the same second temperature, and will not exceed a predetermined threshold. Alternatively, the difference between the relative signal obtained at the second temperature before and after PCR and the relative signal obtained at the first temperature before and after PCR can be compared to a predetermined threshold to determine the presence of a cleaved second LOCS; wherein in the presence of an open second LOCS, the difference is greater than the predetermined threshold, and in the absence of an open second LOCS, the difference is less than the predetermined threshold, as demonstrated in Example 15 (Analysis Method C). Detection of each cleaved LOCS reporter indicates the presence of the corresponding target in the sample.

[0623] By way of non-limiting example, another alternative is to measure the height of the melting peak in the melting feature, which is equal to the d fluorescence / d temperature (dF / dT) value at the Tm of each cleaved LOCS. However, the usability of this method is not limited to determining the dF / dT value at the Tm of the cleaved LOCS, but also includes a temperature range that includes Tm. The dF / dT value at the first or second temperature (A ° C) is equal to the fluorescence level gradient across (AN) ° C and (A + N) ° C. As demonstrated in Example 15 (Analysis Method D), the dF / dT value at the first temperature calculated based on the fluorescence signal at (first temperature + N) ° C and (first temperature - N) ° C is higher than a predetermined threshold only in the presence of an open first LOCS. The dF / dT value at the second temperature higher than the first temperature calculated based on the fluorescence signal at (second temperature + N) ° C and (second temperature - N) ° C is higher than a predetermined threshold only in the presence of an open second LOCS. The detection of each cleaved LOCS reporter indicates the presence of the corresponding target gene in the sample. The dF / dT at the first and second temperatures can be expressed as a ratio that is unique for each of the possible combinations of open and LOCS. By determining whether the ratio falls within a predefined range of values, this information can be used to detect specific open LOCS and, therefore, the corresponding target.

[0624] In some embodiments, the fluorescent signal or its derivative at the first, second and / or additional temperature is compared relative to a predetermined threshold value, wherein the signal from the cleaved open first LOCS exceeds the predetermined threshold value at the first temperature and the cleaved open LOCS exceeds the predetermined threshold value at the second temperature, and the intact closed first and second LOCS do not exceed the predetermined threshold value at the first and second temperatures. Therefore, the utility of the predetermined threshold values ​​at the first and second temperatures can be used to detect the cleaved open first and second LOCS. Detection of the cleaved open first and second LOCS indicates the presence of the first and second target genes in the sample. In other embodiments, the fluorescent signal or its derivative at the first, second and / or additional temperature can be expressed as a ratio, wherein there is a unique ratio value for each possible combination of open and / or closed LOCS. These unique ratio values ​​can be used to detect the cleaved open first and second LOCS and indicate the presence of the first and second target genes in the sample.

[0625] In some embodiments, detection, differentiation, and / or quantification of a first and a second target using a first and a second LOCS in a single fluorescent channel is described. However, one skilled in the art will recognize that the methods are applicable to detection, differentiation, and / or quantification of more than two targets, as demonstrated in Example 15.

[0626] Exemplary Applications of LOCS Oligonucleotides

[0627] - Detection of targets during or after target amplification

[0628] The LOCS oligonucleotides of the present invention can be used to determine the presence of amplified target nucleic acid sequences. There are no particular limitations on the amplification techniques to which LOCS reporters can be applied. Amplicons generated by various reactions can be detected by LOCS reporters, provided that the presence of the target amplicon promotes cleavage or degradation of the LOCS reporter to produce an open LOCS structure. Non-limiting examples of methods for cleaving or degrading loop regions contained within LOCS structures include cleavage by MNAzymes, DNA enzymes, ribozymes, restriction enzymes, endonucleases, or degradation by exonucleases (including but not limited to the exonuclease activity of polymerases).

[0629] Typically, nucleic acid amplification techniques utilize an enzyme (e.g., a polymerase) to generate copies of a target nucleic acid that is specifically bound by one or more oligonucleotide primers. Non-limiting examples of amplification techniques that can use LOCS oligonucleotides include one or more of 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 (TMA), self-sustaining sequence replication (3SR), and nucleic acid sequence-based amplification (NASBA).

[0630] Those skilled in the art will readily appreciate that the above applications of LOCS oligonucleotides are provided for non-limiting exemplary purposes only. The disclosed LOCS oligonucleotides can be used in any primer-based nucleic acid amplification technology, and the present invention is not limited to those specifically described embodiments.

[0631] -Detection of amplicons generated using LOCS reporters

[0632] As discussed above, the LOCS reporters of the present invention can be used in any polynucleotide amplification technique, non-limiting examples of which include PCR, RT-PCR, SDA, HDA, RPA, LAMP, RCA, TMA, 3SR, or NASBA.

[0633] The amplicons generated by the technology utilizing LOCS reporters can be cleaved or degraded using any suitable method known in the art. Non-limiting examples include the use of catalytic nucleic acids, exonucleases (see Example 11), endonucleases (see Example 10), and the like.

[0634] MNAzymes can be used to open LOCS reporters by detecting amplicons generated by methods such as PCR, RT-PCR, SDA, HDA, RPA, TMA, LAMP, RCA, 3SR and NASBA. MNAzymes can include one or more partzymes. MNAzymes are multicomponent nucleic acid enzymes that are assembled and are catalytically active only in the presence of an assembly facilitator, which can be, for example, a target to be detected, such as an amplicon generated from a polynucleotide sequence using primers. MNAzymes are composed of multiple part-enzymes (or partzymes) that self-assemble in the presence of one or more assembly facilitators and form active MNAzymes that catalytically modify a substrate. The substrate and assembly facilitator (target) are separate nucleic acid molecules. Partzymes have multiple domains comprising (i) a sensor arm that binds to an assembly facilitator (such as a target nucleic acid), (ii) a substrate arm that binds to a substrate, and (iii) a partial catalytic core sequence that combines after 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 an assembly facilitator, the MNAzyme modifies its substrate. This substrate modification can be linked to signal generation, and thus, the MNAzyme can generate an output signal for enzyme amplification. The assembly facilitator can be a target nucleic acid present in a biological or environmental sample (e.g., an amplicon generated from a polynucleotide target using primers). In this case, detection of modification of the substrate 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 contents of each of these documents are incorporated herein by reference in their entirety).

[0635] -Diagnostic applications

[0636] According to the methods described herein, LOCS oligonucleotides can be used for diagnostic and / or prognostic purposes. Diagnostic and / or prognostic methods can be performed ex vivo or in vitro. However, the methods of the present invention are not necessarily for diagnostic and / or prognostic purposes, and therefore non-diagnostic or prognostic applications are also contemplated.

[0637] In some embodiments, the methods described herein can be used to diagnose an infection in a subject. For example, the methods can be used to diagnose an infection in a subject with bacteria, viruses, fungi / yeasts, protozoa, and / or nematodes. In one embodiment, the virus can be an enterovirus. The subject can be a cattle, horse, sheep, primate, bird, or rodent. For example, the subject can be a mammal, such as a human, dog, cat, horse, sheep, goat, or cattle. The subject may suffer from a disease caused by an infection. For example, the subject may suffer from meningitis caused by an enterovirus infection. Therefore, in certain embodiments, the methods of the present invention can be used to diagnose meningitis.

[0638] The method of the present invention can be performed on a sample. The sample can be derived from any source. For example, the sample can be obtained from an environmental source, an industrial source, or by chemical synthesis.

[0639] It will be understood that a "sample" as contemplated herein encompasses a sample that has been modified from its original state, for example by purification, dilution or the addition of any other component or components.

[0640] The methods of the present invention, including but not limited to diagnostic and / or prognostic methods, can be performed on biological samples. The biological sample can be taken from a subject. Stored 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, feces, saliva, lymph, bile, sputum, tears, cerebrospinal fluid, bronchoalveolar lavage fluid, synovial fluid, semen, ascites, breast milk, and pus.

[0641] Reagent test kit

[0642] The present invention provides kits comprising one or more reagents for performing the methods of the present invention. Typically, the kits for practicing the methods of the present invention contain all necessary reagents for practicing the methods.

[0643] In some embodiments, the kit may include oligonucleotide components capable of forming an MNAzyme in the presence of an appropriate 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 the self-assembly of the first and second partzymes with the substrate into the MNAzyme requires association with an assembly facilitator (e.g., an amplicon) present in the test sample. Thus, in such embodiments, the first and second partzymes and the LOCS oligonucleotide comprising the substrate within the loop region may be applied to the test sample in order to determine the presence of one or more target amplicons. Typically, the kit includes at least one LOCS oligonucleotide as provided herein.

[0644] Typically, the kits of the present invention will also include other reagents, washing reagents, enzymes and / or other reagents required for performing the methods of the present invention (such as PCR or other nucleic acid amplification techniques).

[0645] The kit may be a fragmentation kit or a combined kit as defined herein.

[0646] The fragmentation kit includes reagents contained in separate containers and can include small glass containers, plastic containers, or plastic or paper strips. Such containers can allow for efficient transfer of reagents from one compartment to another while avoiding cross-contamination of samples and reagents, and allow for the addition of reagents or solutions from each container from one compartment to another in a quantitative manner.

[0647] Such a kit may also include a container for receiving a test sample, a container containing reagents used in the assay, multiple containers containing wash reagents, and multiple containers containing detection reagents.

[0648] The combined kit includes all components of the reaction assay in a single container (eg, in a single box containing each of the desired components).

[0649] The kits of the present invention may also include instructions for performing appropriate methods using the kit components.The kits and methods of the present invention may be used in conjunction with automated analytical equipment and systems, including, but not limited to, real-time PCR machines.

[0650] For applications in amplification, detection, identification or quantification of different targets, a single kit of the invention may be used, or alternatively, different kits may be required, e.g., containing reagents specific for each target. The methods and kits of the invention may be used in any situation where it is desired to detect, identify or quantify any entity.

[0651] It will be appreciated by those skilled in the art that various changes and / or modifications may be made to the invention as disclosed in the specific embodiments without departing from the spirit or scope of the invention as broadly described. Therefore, the present embodiments are to be considered in all respects as illustrative and not restrictive.

[0652] Examples

[0653] The present invention will now be further described in greater detail by reference to the following specific examples, which should not be construed in any way as limiting the scope of the present invention.

[0654] Example 1: Using LOCS reporters to improve multiplexing capabilities

[0655] In the following example, LOCS reporters are used to increase the number of targets that can be detected from a single fluorescence channel. In this example, two LOCS reporters are labeled 5' with a fluorophore (FAM) and 3' with a quencher. The loop region of the LOCS reporter contains the nucleic acid substrate, and the stem region contains a series of complementary base pairs that confine the LOCS reporter in a stem-loop configuration. In this configuration, the fluorophore and quencher are in close proximity and quench fluorescence in the absence of target.

[0656] Oligonucleotides

[0657] Oligonucleotides specific for this experiment include: LOCS-1 (SEQ ID NO: 1), LOCS-2 (SEQ ID NO: 2), partzyme A1 (SEQ ID NO: 3), partzyme B1 (SEQ ID NO: 4), partzyme A2 (SEQ ID NO: 5), partzyme B2 (SEQ ID NO: 6), forward primer 1 (SEQ ID NO: 7), reverse primer 1 (SEQ ID NO: 8), forward primer 2 (SEQ ID NO: 9), and reverse primer 2 (SEQ ID NO: 10). The sequences are listed in the sequence listing. The oligonucleotides specific for MgPa amplification and detection are LOCS-1, partzyme A1, partzyme B1, forward primer 1, and reverse primer 1. The oligonucleotides specific for TV-Btub amplification and detection are LOCS-2, partzyme A2, partzyme B2, forward primer 2, and reverse primer 2.

[0658] Reaction conditions

[0659] use CFX96 thermal cycler performs real-time amplification and detection of target sequences in a total reaction volume of 20 μL. Cycling parameters are: 95°C for 2 minutes, 95°C for 5 seconds and 61°C for 30 seconds for 10 touchdown cycles (0.5°C decrement for each cycle) and 95°C for 5 seconds and 52°C for 40 seconds for 40 cycles (data collected at 52°C steps). Melting curve parameters are 0.5°C increments from 20°C to 90°C for 5 seconds (keeping data acquisition). All reactions are repeated and contain 40nM of each forward primer, 200nM of each reverse primer, 200nM of each partenzyme A, 200nM of each partenzyme B, 200nM of each LOCS reporter, 2mM MgCl2 (Bioline) and 1x SensiFAST probe No-ROX mixture (Bioline). The reactions contained G-Block templates homologous to the MgPa and / or TV-Btub genes (10,000 or 40 copies), or no target (nuclease-free H2O (NF H2O)).

[0660] result

[0661] Using an in vitro target amplification method known as PCR, two MNAzymes (MNAzyme 1 and MNAzyme 2) were used to monitor the amplification of the target nucleic acid in real time by cleaving their corresponding LOCS reporters (LOCS-1 and LOCS-2, respectively). MNAzyme 1 was designed to detect sequences homologous to the MgPa gene (Mycoplasma genitalium) and cleave and open LOCS-1; and MNAzyme 2 was designed to detect sequences homologous to TV-Btub (Trichomonas vaginalis) and cleave and open LOCS-2. In this experiment, amplification and detection were performed in a single tube containing all MNAzymes, primers, and LOCS oligonucleotides. The presence of MgPa or TV-Btub, or both MgPa and TV-Btub (indicating a sample with a co-infection) was detected by an increase in signal in the FAM channel.

[0662] Figure 4 The results shown demonstrate the effects of the MgPa Figure 4 A) TV-Btub( Figure 4 C) or both MgPa and TV-Btub ( Figure 4 E) Plots of the corresponding PCR amplifications obtained in reactions with 10,000 copies (black line), 40 copies (grey line), or 0 copies (dashed line) of the target MgPa ( Figure 4 B) TV-Btub( Figure 4 D) or both MgPa and TV-Btub ( Figure 4 F) shows the melting curve characteristics obtained after amplification from a reaction containing 10,000 copies (black line) and 40 copies (grey line). The result is the average value of the repeated reactions drawn using Microsoft Excel (version 14). The data from this example proves that the LOCS melting characteristics (Tm=35°C and 41°C) produced in the presence of the MgPa gene target are different from the LOCS melting characteristics (Tm=50°C) produced in the presence of the TV-Btub gene target. Further, the LOCS melting characteristics in the presence of both the MgPa gene target and the TV-Btub gene target are also different from the above-mentioned melting characteristics in the presence of only a single gene target. When both targets are present, the melting curve characteristics (Tm=35°C, 41°C and 50°C) indicate that both targets are detected.

[0663] The data from this experiment also demonstrated that the melt profile of each open LOCS was reproducible for reactions containing high target concentrations (10,000 gene copies) and low target concentrations (40 gene copies). This example demonstrates that two targets co-amplified in a single well using a single fluorescent channel can be distinguished based on the unique LOCS melt profile. The example provides a simple method that can be used to detect multiple targets in a single well using a single fluorescent channel.

[0664] The melting temperature (Tm) of the complete closed LOCS reporter was designed to be above the annealing temperature of the reaction (52°C) so that these LOCS remain fully quenched during reactions without target amplification. The Tm of the stem region was designed to be unique for each LOCS reporter and this was achieved by modifying the length and nucleotide composition of the complementary region that forms the stem. In this example, the data is consistent with the use of an MNAzyme to cleave the loop region, resulting in the opening of the LOCS, allowing separation into two fragments. Due to the large difference in Tm between the closed complete LOCS reporter and the open LOCs reporter, the open LOCS fragment was not stable enough to maintain duplex formation at the annealing and data acquisition temperatures, thus generating a fluorescent signal.

[0665] Real-time monitoring of the fluorescence curve generated by two target genes that exceed any threshold value has produced a value that can be called Ct (cycle threshold). The fluorescence curve indicated by observing during the amplification phase is present in a given sample as one or two target genes in the target gene. However, the identity of a specific target can not be distinguished using only the amplification curve. In this case, melting curve analysis is performed to determine which LOCS reporter has been cracked, and one or more specific gene targets present in the identification sample are identified as such. After the amplification phase, the sample is exposed to a temperature gradient, in which different melting curve characteristics correspond to different universal stems. Due to the Tm difference between open and complete LOCS reporters, compared with uncracked closed LOCS, the LOCS reporter stem of cracking can dissociate at a lower temperature. In addition, due to different stem lengths and base pair compositions, the different stems (stem 1 and stem 2) in LOCS-1 and LOCS-2 have produced unique fluorescence melting curve characteristics.

[0666] Example 2: Using the same LOCS reporter to detect various gene targets

[0667] In this example, two LOCS reporters (LOCS-1 and LOCS-2) were used to distinguish between various genetic targets, demonstrating its versatility and applicability to analyzing any target of interest. Using PCR amplification, several MNAzymes (MNAzymes 1-6) were used to detect their specific gene targets and cleave their corresponding LOCS reporters (LOCS-1 and LOCS-2). MNAzymes 1, 3, and 4 each had the ability to cleave LOCS-1 in the presence of their corresponding gene targets. MNAzymes 2, 5, and 6 each had the ability to cleave LOCS-2 in the presence of their corresponding gene targets. In this example, the duplex combinations summarized in Table 1 were demonstrated, where amplification and detection were performed simultaneously in a single tube.

[0668] Oligonucleotides

[0669] Oligonucleotides specific for this experiment include: LOCS-1 (SEQ ID NO: 1), LOCS-2 (SEQ ID NO: 2), partzyme A1 (SEQ ID NO: 3), partzyme B1 (SEQ ID NO: 4), partzyme A2 (SEQ ID NO: 5), partzyme B2 (SEQ ID NO: 6), forward primer 1 (SEQ ID NO: 7), reverse primer 1 (SEQ ID NO: 8), forward primer 2 (SEQ ID NO: 9), reverse primer 2 (SEQ ID NO: 10), partzyme A3 (SEQ ID NO: 11), partzyme B3 (SEQ ID NO: 12), partzyme A4 (SEQ ID NO: 13), partzyme B4 (SEQ ID NO: 14), forward primer 3 (SEQ ID NO: 15), reverse primer 3 (SEQ ID NO: 16), forward primer 4 (SEQ ID NO: 17), reverse primer 4 (SEQ ID NO: 18), partzyme A5 (SEQ ID NO: 19), partzyme B5 (SEQ ID NO: 20). NO: 20), partzyme A6 (SEQ ID NO: 21), partzyme B6 (SEQ ID NO: 22), forward primer 5 (SEQ ID NO: 23), reverse primer 5 (SEQ ID NO: 24), forward primer 6 (SEQ ID NO: 25), and reverse primer 6 (SEQ ID NO: 26). The sequences are listed in the sequence listing.

[0670] Table 1: Reaction components of Example 2 PCR mixture AF

[0671]

[0672] Reaction components and conditions.

[0673] use CFX96 thermal cycler performs real-time amplification and detection of target sequences in a total reaction volume of 20 μL. Cycling parameters are: 95°C for 2 minutes, 95°C for 5 seconds and 61°C for 30 seconds for 10 touchdown cycles (0.5°C decrement per cycle) and 95°C for 5 seconds and 52°C for 40 seconds for 40 cycles (data collected at 52°C step). Melting curve parameters are 0.5°C increments from 20°C to 90°C for 5 seconds (maintaining data acquisition). All reactions are performed in duplex reactions containing the oligonucleotides specified in Table 1. Each reaction contains 40nM of each forward primer, 200nM of each reverse primer, 200nM of each partenzyme A, 200nM of each partenzyme B, 200nM of each LOCS reporter, 2mMMgCl2 (Bioline) and 1x SensiFAST probe No-ROX mixture (Bioline). Reactions contained G-Block template (10,000 or 40 copies) or no target (NF H2O). Figure 5 and 6 The results presented in are the means of replicates plotted using Microsoft Excel (version 14).

[0674] result

[0675] Figure 5 The results shown in (above) demonstrate the effect of the MgPa( Figure 5 A1)、HMPV( Figure 5 B1) and CT-ompA( Figure 5 The results shown in the figure below are plots of PCR amplification obtained from reactions containing 10,000 copies (black line), 40 copies (grey line), or 0 copies (dashed line) of MgPa ( Figure 5 A2)、HMPV( Figure 5 B2) and CT-ompA( Figure 5 C2) Melting curve characteristics obtained from reactions with 10,000 copies (black line) and 40 copies (grey line) of a gene target. These three targets were specifically detected using MNAzymes 1, 3, and 5; each of which has a target-specific sensor arm, but all three targets have the same substrate binding arm. Once formed in the presence of its target assembly facilitator, each MNAzyme is able to bind, cleave, and open the same universal LOCS-1 comprising a first universal substrate and a first universal stem. Thus, the presence of each target produces a melting curve with a peak at 40°C, corresponding to the Tm of the first universal stem.

[0676] Figure 6 The results shown in (top) demonstrate the targeting of the target TV-Btub ( Figure 6 A1)、VZV( Figure 6 B1) and rpoB( Figure 6 The results shown in the figure below are plots of PCR amplification obtained from reactions containing 10,000 copies (black line), 40 copies (grey line), or 0 copies (dashed line) of TV-Btub ( Figure 6 A2)、VZV( Figure 6 B2) and rpoB( Figure 6 C2) Melting curve characteristics obtained from reactions with 10,000 copies (black line) and 40 copies (grey line) of a gene target. These three targets were specifically detected using MNAzymes 2, 4, and 6; each of which has a target-specific sensor arm, but all three targets have the same substrate binding arm. Once formed in the presence of its target assembly facilitator, each MNAzyme is able to bind, cleave, and open the same universal LOCS-2 comprising a second universal substrate and a second universal stem. Thus, the presence of each target produces a melting curve with a peak at 50°C, corresponding to the Tm of the second universal stem.

[0677] The data from this example demonstrates that using the same universal LOCS reporter yields comparable melting profiles for detecting different target genes. LOCS melting curves have been shown to be independent of the target sequence and can therefore be easily implemented in any assay. This example demonstrates that the LOCS reporter is universal and can be used to detect any desired target gene or transcript.

[0678] Example 3: Generating LOCS reporters with different melting temperatures using different stems to improve multiplexing capabilities

[0679] In this example, LOCS reporters comprising different stem components were used to demonstrate the effect of stem length and base pair composition on the final melting characteristics. In the current example, four LOCS reporters were labeled 5' with a fluorophore (FAM) and 3' with a quencher. LOCS-1 and LOCS-3 contained the same loop region comprising substrate 1; however, the stem region of LOCS-3 (stem 3) was a single base pair longer than that of LOCS-1 (stem 1), resulting in a structure with a slightly higher predicted Tm in both the intact (closed) and open states. Isothermal signal detection was monitored using an MNAzyme with a target sensor arm capable of guiding assembly in the presence of a target assembly facilitator (AF-CT-Cds) and a substrate arm capable of binding and cleaving substrate 1 within the loop of LOCS-1 or LOCS-3.

[0680] Similarly, LOCS-2 and LOCS-4 contain the same loop region comprising substrate 2; however, the stem region of LOCS-2 (stem 2) is two base pairs longer than that of LOCS-4 (stem 4), resulting in a structure with a higher predicted Tm in both the intact (closed) and open states. PCR amplification was monitored using an MNAzyme with a target sensor arm capable of directing assembly in the presence of a target assembly facilitator (AF-TFRC) and a substrate arm capable of binding and cleaving substrate 2 within the loop of either LOCS-2 or LOCS-4.

[0681] - Reaction components and conditions

[0682] Oligonucleotides specific for this experiment include: LOCS-1 (SEQ ID NO: 1), LOCS-2 (SEQ ID NO: 2), LOCS-3 (SEQ ID NO: 27), LOCS-4 (SEQ ID NO: 28), partzyme A7 (SEQ ID NO: 29), partzyme B7 (SEQ ID NO: 30), partzyme A8 (SEQ ID NO: 31), partzyme B8 (SEQ ID NO: 32), AF-CT-Cds (SEQ ID NO: 33), and AF-TFRC (SEQ ID NO: 38). The sequences are listed in the sequence listing.

[0683] Real-time detection of target sequences was performed in a total reaction volume of 20 μL. Each reaction contained 1x NH4 buffer (Bioline), 8 mM MgCl2 (Bioline), 200 nM of each partzyme, and 200 nM of the LOCS reporter. Reactions contained target assembly facilitator at a final concentration of 10 nM or lacked target (no DNA control). Reactions were placed in Incubation was performed on a CFX96 thermal cycler at 52°C with acquisition every 10 seconds for a total of 150 cycles. Next, the samples were subjected to an increasing temperature gradient from 20°C to 90°C in 0.5°C increments with a 5 second hold time (acquisition during each hold). Figure 7 The results presented in are the means of replicates plotted using Microsoft Excel (version 14).

[0684] result

[0685] Table 2 and Figure 7 The results shown illustrate the difference in melting curve characteristics obtained for a closed, intact LOCS reporter present in a reaction lacking target (dashed line) and a melting curve characteristic consistent with an open LOCS reporter (black line) in a reaction containing a target directing the assembly of an MNAzyme capable of cleaving and opening a specific LOCS reporter.

[0686] Table 2: Figure 7 Summary of the melting temperatures (Tm) of intact (closed) and open LOCS reporters shown in (Panels A, B, C, and D).

[0687]

[0688] Further, the data from this example show the differences in the melting characteristics obtained when the sequence and / or length of the stem region are changed. The data show that the melting characteristics produced by open LOCS-1 have a lower Tm (30°C) compared to open LOCS-3 (36°C). Similarly, the melting characteristics produced by open LOCS-4 have a lower Tm (32°C) compared to open LOCS-2 (50°C). These results are consistent with the fact that the stems of LOCS-1 and LOCS-4 are shorter than those of LOCS-3 and LOCS-2, respectively. In addition, each LOCS generates a unique melting characteristic that is different from other LOCS reporters. This experiment proves that by changing the length and composition of the stem region, different melting characteristics can be produced. Therefore, a group of LOCS reporters can be used simultaneously to detect multiple targets from a single channel. In addition, the data generated in this experiment were obtained using isothermal target detection, which illustrates that LOCS reporters can be used together with a variety of different biosensing technologies.

[0689] Example 4: LOCS reporter compatibility across several platforms

[0690] In this example, the compatibility of two LOCS reporters across several commonly used PCR platforms was tested. Two MNAzymes (MNAzyme 1 and MNAzyme 2) were used to monitor the amplification of target nucleic acids in real time by cleaving their corresponding LOCS reporters (LOCS-1 and LOCS-2, respectively). In this example, amplification and detection of two different genes, namely the MgPa gene (Mycoplasma genitalium) and TV-Btub (Trichomonas vaginalis), were performed simultaneously in a single tube, both of which were capable of generating a signal in the FAM channel.

[0691] Reaction components and conditions

[0692] Oligonucleotides specific for this experiment include: LOCS-1 (SEQ ID NO: 1), LOCS-2 (SEQ ID NO: 2), partzyme A1 (SEQ ID NO: 3), partzyme B1 (SEQ ID NO: 4), partzyme A2 (SEQ ID NO: 5), partzyme B2 (SEQ ID NO: 6), forward primer 1 (SEQ ID NO: 7), forward primer 2 (SEQ ID NO: 8), reverse primer 1 (SEQ ID NO: 9), and reverse primer 2 (SEQ ID NO: 10). The oligonucleotides specific for MgPa amplification and detection are LOCS-1, partzyme A1, partzyme B1, forward primer 1, and reverse primer 1.

[0693] The oligonucleotides specific for TV-Btub amplification and detection are LOCS-2, partzyme A2, partzyme B2, forward primer 2, reverse primer 1, and reverse primer 2. The sequences are listed in the sequence listing.

[0694] The cycling parameters were: 95°C for 2 min, 10 touchdown cycles at 95°C for 5 sec and 61°C for 30 sec (0.5°C decrement per cycle), and 40 cycles at 95°C for 5 sec and 52°C for 40 sec (data collected at the 52°C step). Melting curve parameters on the CFX96 thermal cycler were 0.5°C increments from 20°C to 90°C, held for 5 seconds (holding data acquisition). Melting curve parameters on the Lightcycler 480 thermal cycler were set to continuous acquisition mode with a ramp rate of 0.02°C for each 20°C to 90°C range and a hold of 1 second at each temperature. Melting curve parameters on the ABI 7500 thermal cycler were set to a ramp rate of 1% from 20°C to 90°C, held for 5 seconds (holding data acquisition). Melting curve parameters on the XxpressPCR thermal cycler were 0.5°C increments from 30°C to 90°C, held for 0.5 seconds (holding data acquisition). All reactions were performed in duplicate 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 LOCS reporter, 2 mM MgCl2 (Bioline) and 1x SensiFAST probe No-ROX mix (Bioline). Real-time amplification and detection of target sequences were performed in a total reaction volume of 20 μL, except for reactions performed on an XXpress PCR thermal cycler in a volume of 15 μL. Reactions contained G-Block template (10,000 or 40 copies) or no target (NF H2O). The CFX96 Thermal Cycler, Lightcycler 480 Thermal Cycler, ABI 7500 Thermal Cycler, and XXpress PCR Thermal Cycler perform real-time amplification and detection of target sequences.

[0695] -result

[0696] Figure 8 and Figure 9 The results in the figure show that when Melting curves obtained after amplification of MgPa and TV-Btub targets when amplification was performed on a CFX96 Thermocycler (Panel A), a Lightcycler 480 Thermocycler (Panel B), an ABI 7500 Thermocycler (Panel C), or an XXpress PCR Thermocycler (Panel D). Both targets were monitored in the FAM channel on all machines. Figure 8 D and Figure 9 The results shown in Figure 4 were obtained using a 3-minute melt curve protocol, thus demonstrating that differentiation of melt curves using LOCS reporters can be achieved using rapid conditions. The data demonstrate the ability to generate comparable melt curve profiles across different platforms using two different LOCS reporters. Further, the data demonstrate the ability to generate comparable melt curve profiles using different melt curve parameters.

[0697] Example 5: LOCS reporter compatibility across several fluorescence channels

[0698] In this example, ten LOCS reporters were tested for compatibility across several commonly used fluorescence channels. Two MNAzymes (MNAzyme 1 and MNAzyme 2) were used to monitor the amplification of target nucleic acids in real time by cleaving their corresponding LOCS reporters. In this example, the same LOCS reporter sequence was used, but the LOCS were labeled with different fluorophore and quencher pairs. In this example, amplification and detection of two different genes, the MgPa gene (Mycoplasma genitalium) and TV-Btub (Trichomonas vaginalis), were performed simultaneously in a single test tube, both capable of generating signal in the same channel. Several fluorescence channels were tested, including FAM, HEX, Texas Red, and Cy5.

[0699] Reaction components and conditions

[0700] Oligonucleotides specific for this experiment include: LOCS-1 (SEQ ID NO:1), LOCS-2 (SEQ ID NO:2), partzyme A1 (SEQ ID NO:3), partzyme B1 (SEQ ID NO:4), partzyme A2 (SEQ ID NO:5), partzyme B2 (SEQ ID NO:6), forward primer 1 (SEQ ID NO:7), forward primer 2 (SEQ ID NO:8), reverse primer 1 (SEQ ID NO:9), reverse primer 2 (SEQ ID NO:10), LOCS-5 (SEQ ID NO:35), LOCS-6 (SEQ ID NO:36), LOCS-7 (SEQ ID NO:37), LOCS-8 (SEQ ID NO:38), LOCS-9 (SEQ ID NO:39), and LOCS-10 (SEQ ID NO:40). Oligonucleotides specific for MgPa amplification and detection are partzyme A1, partzyme B1, forward primer 1, reverse primer 1, and LOCS-1, LOCS-5, LOCS-7, or LOCS-9. Oligonucleotides specific for TV-Btub amplification and detection are partzyme A2, partzyme B2, forward primer 2, reverse primer 2, and LOCS-2, LOCS-6, LOCS-8, or LOCS-10. Sequences are listed in the sequence listing.

[0701] The cycling parameters were: 95°C for 2 min, 10 touchdown cycles at 95°C for 5 sec and 61°C for 30 sec (0.5°C decrement per cycle), and 40 cycles at 95°C for 5 sec and 52°C for 40 sec (data collected at the 52°C step). Melting curve parameters on a CFX96 thermal cycler were 0.5°C increments from 20°C to 90°C for 5 seconds (hold data acquisition). All reactions were performed in duplicate 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 LOCS reporter, 2 mM MgCl2 (Bioline), and 1x SensiFAST probe No-ROX mix (Bioline). A CFX96 thermal cycler performs real-time amplification and detection of target sequences in a total reaction volume of 20 μL. Reactions contain G-Block template (10,000 or 40 copies) or no target (NF H2O).

[0702] -result

[0703] Figure 10 The results show that in Melting curves obtained after amplification of the MgPa and TV-Btub targets, respectively, when amplification was performed on a CFX96 thermal cycler. Both targets were monitored simultaneously in a single fluorescence channel. The results shown in black demonstrate the melting curve obtained in the presence of the MgPa gene, and the results shown in gray demonstrate the melting curve characteristics obtained in the presence of the TV-Btub target. The solid line represents a target concentration of 10,000 copies, and the dashed line represents a target concentration of 40 copies. Figure 10 The results shown in panel A show melting curves obtained using LOCS-1 and LOCS-2 in the FAM channel. Figure 10 The melting curves shown in B were obtained using LOCS-5 and LOCS-6 in the HEX channel. Figure 10 The melting curves shown in C were obtained using LOCS-7 and LOCS-8 in the Texas Red channel, and Figure 10 Melting curves shown in D were obtained using LOCS-9 and LOCS-10 in the Cy5 channel. The data demonstrate the ability to generate comparable melting curve characteristics across different fluorescence channels using the same two LOCS stems and substrates but simply changing the fluorophore-quencher pair.

[0704] Example 6: Simultaneous detection of three targets in a single fluorescence channel using three LOCS reporters

[0705] In the following example, LOCS reporters are used to increase the number of targets that can be detected from a single fluorescence channel. In this example, three LOCS reporters are labeled 5' with a fluorophore (FAM) and 3' with a quencher. The loop region of the LOCS reporter contains the nucleic acid substrate, and the stem region contains a series of complementary base pairs that confine the LOCS reporter in a loop-stem configuration. In this configuration, the fluorophore and quencher are in close proximity and quench fluorescence in the absence of target.

[0706] Oligonucleotides

[0707] Oligonucleotides specific for this experiment include: LOCS-1 (SEQ ID NO: 1), LOCS-2 (SEQ ID NO: 2), partzyme A1 (SEQ ID NO: 3), partzyme B1 (SEQ ID NO: 4), partzyme A2 (SEQ ID NO: 5), partzyme B2 (SEQ ID NO: 6), forward primer 1 (SEQ ID NO: 7), reverse primer 1 (SEQ ID NO: 8), forward primer 2 (SEQ ID NO: 9), reverse primer 2 (SEQ ID NO: 10), LOCS-11 (SEQ ID NO: 45), partzyme A9 (SEQ ID NO: 43), partzyme B9 (SEQ ID NO: 44), forward primer 7 (SEQ ID NO: 41), and reverse primer 7 (SEQ ID NO: 42). The sequences are listed in the sequence listing. The oligonucleotides specific for MgPa amplification and detection are LOCS-1, partzyme A1, partzyme B1, forward primer 1, and reverse primer 1. The oligonucleotides specific for amplification and detection of TV-Btub are LOCS-2, partzyme A2, partzyme B2, forward primer 2, and reverse primer 2. The oligonucleotides specific for amplification and detection of CTcry are LOCS-11, partzyme A9, partzyme B9, forward primer 7, and reverse primer 7.

[0708] Reaction conditions

[0709] use CFX96 thermal cycler performs real-time amplification and detection of target sequences in a total reaction volume of 20 μL. Cycling parameters are: 95°C for 2 minutes, 95°C for 5 seconds and 61°C for 30 seconds for 10 touchdown cycles (0.5°C decrement per cycle) and 95°C for 5 seconds and 52°C for 40 seconds for 35 cycles (data collected at 52°C steps). Melting curve parameters are 0.5°C increments from 20°C to 90°C for 5 seconds (keeping data acquisition). All reactions are repeated and contain 40nM of each forward primer, 200nM of each reverse primer, 200nM of each partenzyme A, 200nM of each partenzyme B, 200nM of each LOCS reporter, 2mM MgCl2 (Bioline) and 1x SensiFAST probe No-ROX mixture (Bioline). The reactions contained G-Block templates homologous to the MgPa and / or TV-Btub and / or CTcry genes (10,000 copies), or no target (NF H2O).

[0710] result

[0711] Using an in vitro target amplification method known as PCR, three MNAzymes (MNAzyme 1, MNAzyme 2, and MNAzyme 9) were used to monitor the amplification of target nucleic acids in real time by cleaving their corresponding LOCS reporters (LOCS-1, LOCS-2, and LOCS-11, respectively). MNAzyme 1 was designed to detect sequences homologous to the MgPa gene (Mycoplasma genitalium) and cleave and open LOCS-1; MNAzyme 2 was designed to detect sequences homologous to TV-Btub (Trichomonas vaginalis) and cleave and open LOCS-2; and MNAzyme 9 was designed to detect sequences homologous to CTcry (Chlamydia trachomatis) and cleave and open LOCS-11. In this experiment, amplification and detection were performed in a single tube containing all MNAzymes, primers, and LOCS oligonucleotides. The presence of MgPa, TV-Btub, CTcry, or various combinations of the three (indicating samples with multiple infections) was detected by an increase in signal in the FAM channel.

[0712] Figure 11 and Figure 12 The results shown illustrate that after amplification, the target MgPa ( Figure 11 A) TV-Bthub( Figure 11 B) CTcry Figure 11 C), MgPa and TV-Bthub ( Figure 11 D), MgPa and CTcry ( Figure 11 E), TV-Bthub and CTcry ( Figure 11 F) or all three MgPa, TV-Bthub and CTcry ( Figure 12 The corresponding melting curve characteristics were obtained from 10,000 replicate reactions of 10,000 replicates. The results are the average of the replicate reactions and plotted using Microsoft Excel (version 14).

[0713] The data from this example, summarized in Table 3, demonstrate that the LOCS melting profile generated in the presence of the MgPa gene target (Tm = 39°C and 66°C) is different from the LOCS melting profile generated in the presence of the TV-Btub (Tm = 49°C and 64°C) and CTcry (Tm = 30°C, 41°C, and 64°C) gene targets. Furthermore, in the presence of two or more gene targets, the LOCS melting profile also differs from the above-described melting profile in the presence of only a single gene target. When both the MgPa and TV-Btub targets are present in a single reaction, the unique melting curve profile (Tm = 39°C, 49°C, and 66°C) indicates that both targets are detected. Similarly, when MgPa and CTcry or TV-Btub and CTcry were present in a single reaction, unique melting curve features ((Tm = 31°C, 41°C, and 74°C) and (Tm = 30°C, 41°C, 49°C, and 66°C), respectively) indicated which two targets were detected. When MgPa, TV-Btub, and CTcry targets were present in a single reaction, unique melting curve features (Tm = 30°C, 41°C, and 49°C) indicated that all three targets were detected.

[0714] Table 3: Figure 11 and 12 Summary of the melting temperatures (Tm) of the LOCS reporters in the presence of one, two, or three targets is shown.

[0715]

[0716] This example demonstrates that three targets co-amplified in a single well using a single fluorescence channel can be distinguished based on a unique LOCS melting signature. The example provides a simple method that can be used to detect multiple targets in a single well using a single fluorescence channel. Due to different stem lengths and base pair compositions, the different stems (stem 1, stem 2, and stem 3) within LOCS-1, LOCS-2, and LOCS-11 produce unique fluorescent melting curve signatures.

[0717] In this particular example, when both MgPa and CTcry targets were present, the Tm values ​​of open LOCS 1 (one peak at approximately 39°C indicating MgPa) and open LOCS 3 (two peaks at approximately 30 / 31°C and approximately 41°C indicating CTcry) resulted in merged peaks. However, the Tm peak of open LOCS 1 (approximately 64°C) can be used to distinguish the reaction containing MgPa, TV-Btub, and CTcry. Figure 12 ) and the reaction containing only TV-Btub and CTcry ( Figure 11 Although merged peaks are not ideal for this method, alternative stem sequences that produce more clearly distinguished peaks can be readily identified using the stem screening assay described in Example 7.

[0718] Example 7: Method for screening sequences suitable for use as universal stems that can be incorporated into LOCS reporter oligonucleotides

[0719] In the following examples, a series of double-stranded oligonucleotides (DSOs) were screened for suitability as universal stems. In the presence of SYBR green I intercalating dye, these DSOs that were not connected by rings were subjected to gradually increased temperature. This melting curve screening assay can be used to check various sequence lengths and composition, to identify a series of DSOs that melt at discrete temperatures and can subsequently be incorporated into LOCS reporter oligonucleotides as universal stems. SYBR green I is an intercalating dye that can produce fluorescent signals when combined with double-stranded DNA structures. After being exposed to the temperature gradient that increases, two oligonucleotide chains of DSO dissociate, and fluorescence decay. The following examples demonstrate that the temperature (Tm) that two oligonucleotides dissociate can be regulated by changing stem length and composition. In addition, DSO was tested under various concentrations to further manipulate Tm by demonstrating how to modify oligonucleotide concentration.

[0720] Oligonucleotides

[0721] Oligonucleotides specific for this experiment include: DSO-1A (SEQ ID NO: 56), DSO-1B (SEQ ID NO: 57), DSO-2A (SEQ ID NO: 58), DSO-2B (SEQ ID NO: 59), DSO-3A (SEQ ID NO: 60), DSO-3B (SEQ ID NO: 61), DSO-4A (SEQ ID NO: 62), DSO-4B (SEQ ID NO: 63), DSO-5A (SEQ ID NO: 64), DSO-5B (SEQ ID NO: 65), DSO-6A (SEQ ID NO: 66), DSO-6B (SEQ ID NO: 67), DSO-7A (SEQ ID NO: 68), DSO-7B (SEQ ID NO: 69), DSO-8A (SEQ ID NO: 70), and DSO-8B (SEQ ID NO: 71). The sequences are listed in the sequence listing.

[0722] Reaction conditions

[0723] use Melting curve analysis was performed on a CFX96 thermal cycler in a total reaction volume of 20 μL. Melting curve parameters were 0.5°C increments from 15°C to 70°C, with a 5-second hold (holding data acquisition). All reactions were performed in duplicate and contained 100 nM, 200 nM, or 300 nM of DSO-A and DSO-B oligonucleotides, 1 μM SYBR Green I, 8 mM MgCl2 (Bioline), and 1x NH4 buffer (Bioline).

[0724] result

[0725] The results shown in Table 4 demonstrate the melting temperatures (Tm) obtained when DSO was subjected to an increasing temperature gradient. The results are the average of replicate reactions plotted using Microsoft Excel (Version 14).

[0726] This example proves that the stem length and GC content of increase can cause higher Tm.In addition, the oligonucleotide concentration that increases also can cause Tm slightly higher, and wherein the difference that observes when concentration increases from 100nM to 200nM is maximum.The method described in this example can be used as screening method, to better identify the alternative stem sequence that produces the peak that more clearly distinguishes.

[0727] Table 4: Summary of Tm of DSO at different concentrations

[0728]

[0729]

[0730] The method can also be used to examine the effects of other components of the reaction mixture, including but not limited to salt concentration, Mg concentration, buffer, dNTP concentration, and other additives. Using this screening method, a series of DSOs can be identified that, when incorporated into LOCS, result in a well-separated Tm ladder. In an alternative format, the DSOs can be labeled with different fluorophore and quencher dye pairs to investigate whether different dye combinations can affect the Tm of a particular DSO.

[0731] Example 8: Simultaneous detection of four targets in a single well using two fluorescent channels and four LOCS reporters utilizing only two stems

[0732] In the following examples, multiple LOCS reporters comprising identical stem compositions and being connected to different substrates (rings) are merged in a single reaction vessel to demonstrate that the identical stem region can be repeatedly used to simultaneously detect and distinguish multiple targets in a single reaction. In the current example, two stems (stem 1 and stem 2) are used to distinguish four different targets across two fluorescent channels. In this example, two LOCS reporters are labeled with 5' (LOCS-12 and LOCS-13) and the other two are labeled with 5' (LOCS-7 and LOCS-8) ​​using the T Texas Red fluorophore. All four LOCS reporters are labeled with 3' quenchers. Each of the four LOCS reporters' ring regions contains different nucleic acid substrates (Sub1, Sub2, Sub3, and Sub4). Two LOCS reporters (one for each fluorophore) contain stem 1 (LOCS-7 and LOCS-12), and the other two LOCS reporters contain stem 2 (LOCS-8 and LOCS-13). The stem region contains a series of complementary base pairs that confine the LOCS reporter to a loop-stem configuration. In this configuration, the fluorophore and quencher are in close proximity, quenching fluorescence in the absence of target. Stem 1 has a lower melting temperature than stem 2 and connects to loops 1 and 3 to detect the targets MgPa and NGoPa at the first detection temperature (40°C), while stem 2 has a higher melting temperature and connects to loops 2 and 4 to distinguish between the targets TV-Btub and gpd at the second detection temperature (50°C).

[0733] Oligonucleotides

[0734] Oligonucleotides specific for this experiment include: LOCS-7 (SEQ ID NO:37), LOCS-8 (SEQ ID NO:38), partzyme A1 (SEQ ID NO:3), partzyme B1 (SEQ ID NO:4), partzyme A2 (SEQ ID NO:5), partzyme B2 (SEQ ID NO:6), forward primer 1 (SEQ ID NO:7), reverse primer 1 (SEQ ID NO:8), forward primer 2 (SEQ ID NO:9), reverse primer 2 (SEQ ID NO:10), LOCS-12 (SEQ ID NO:46), LOCS-13 (SEQ ID NO:47), partzyme A10 (SEQ ID NO:48), partzyme B10 (SEQ ID NO:49), partzyme A11 (SEQ ID NO:50), partzyme B11 (SEQ ID NO:51), forward primer 8 (SEQ ID NO:52), reverse primer 8 (SEQ ID NO:53), forward primer 9 (SEQ ID NO:54), reverse primer 9 (SEQ ID NO:55), The oligonucleotides specific for amplifying and detecting MgPa are LOCS-7, partzyme A1, partzyme B1, forward primer 1, and reverse primer 1. The oligonucleotides specific for amplifying and detecting TV-Btub are LOCS-8, partzyme A2, partzyme B2, forward primer 2, and reverse primer 2. The oligonucleotides specific for amplifying and detecting NGopa are LOCS-12, partzyme A10, partzyme B10, forward primer 8, and reverse primer 8. The oligonucleotides specific for amplifying and detecting gpd are LOCS-13, partzyme A11, partzyme B11, forward primer 9, and reverse primer 9.

[0735] Reaction conditions

[0736] use CFX96 thermal cycler performs real-time amplification and detection of target sequences in a total reaction volume of 20 μL. Cycling parameters are: 95°C for 2 minutes, 95°C for 5 seconds and 61°C for 30 seconds for 10 touchdown cycles (0.5°C decrement per cycle) and 95°C for 5 seconds and 52°C for 40 seconds for 35 cycles (data collected at 52°C steps). Melting curve parameters are 0.5°C increments from 20°C to 90°C for 5 seconds (keeping data acquisition). All reactions are repeated and contain 40nM of each forward primer, 200nM of each reverse primer, 200nM of each partenzyme A, 200nM of each partenzyme B, 200nM of each LOCS reporter, 2mM MgCl2 (Bioline) and 1x SensiFAST probe No-ROX mixture (Bioline). The reactions contained G-Block templates homologous to the MgPa, TV-Btub, NGopa, or gpd genes (10,000 copies) or no target (NF H2O).

[0737] result

[0738] Using an in vitro target amplification method known as PCR, four MNAzymes (MNAzyme 1, MNAzyme 2, MNAzyme 10, and MNAzyme 11) were used to monitor the amplification of target nucleic acids in real time by cleaving their corresponding LOCS reporters (LOCS-7, LOCS-8, LOCS-12, and LOCS-13, respectively). MNAzyme 1 was designed to detect sequences homologous to the MgPa gene (Mycoplasma genitalium) and cleave and open LOCS-7; MNAzyme 2 was designed to detect sequences homologous to the TV-Btub gene (Trichomonas vaginalis) and cleave and open LOCS-8; MNAzyme 10 was designed to detect sequences homologous to the NGopa gene (Neisseria gonorrhoeae) and cleave and open LOCS-12; and MNAzyme 11 was designed to detect sequences homologous to the gpd gene (Herpes simplex virus type 2) and cleave and open LOCS-13. In this experiment, amplification and detection were performed in a single tube containing all MNAzymes, primers, and LOCS oligonucleotides.

[0739] The presence of the MgPa or TV-Btub gene is detected by an increase in signal in the Texas Red channel, and the presence of the NGopa or gpd gene is detected by an increase in signal in the FAM channel. Discrimination of MgPa or TV-Btub in the Texas Red channel and NGopa or gpd in the FAM channel is determined based on unique melting curve features. Figure 13 The results shown in A show the corresponding melting curve characteristics obtained in the Texas Red channel from reactions containing 10,000 copies of the MgPa (black line) or TV-Btub (grey line) gene target after amplification.

[0740] Figure 13 The results shown in Figure B show the corresponding melting curve profiles obtained in the FAM channel from reactions containing 10,000 copies of the NGopa (black line) or gpd (gray line) gene target after amplification. Results are the average of replicate reactions plotted using Microsoft Excel (version 14). The presence of MgPa is determined by an increase in Texas Red signal and a unique melting feature in the Texas Red channel (Tm = 43°C). The presence of TV-Btub is determined by an increase in Texas Red signal and a unique melting feature in the Texas Red channel (Tm = 54°C). The presence of NGopa is determined by an increase in FAM signal and a unique melting feature in the FAM channel (Tm = 26°C and 42°C). The presence of gpd is determined by an increase in FAM signal and a unique melting feature in the FAM channel (Tm = 49°C).

[0741] As summarized in Table 5, the data from this example prove that, by being connected to different substrates (rings) and detecting it across different fluorescence channels, the same general stem region can be repeatedly used in a single reaction. In addition, the data prove that, can distinguish four targets that amplify together in a single hole and use two fluorescence channels to monitor based on unique LOCS melting temperature. The example provides the simple method that can be used for monitoring multiple targets in a single hole. Using the same stem sequence to distinguish multiple targets in a single reaction helps to simply design a highly multiplexed mensuration, and makes the LOCS probe can be quickly and easily applicable to detecting any selected gene target.

[0742] Table 5: Figure 13 Summary of the melting temperatures (Tm) of open LOCS reporters in the presence of one of the four targets shown.

[0743]

[0744] Example 9 - The same stem sequence can be combined with different substrates within the LOCS reporter to produce reproducible melting temperatures

[0745] In the following example, three LOCS reporters are used to demonstrate that the same stem can be paired with different loop sequences (substrates) to produce comparable melting characteristics. In this example, all three LOCS reporters are labeled 5' with a FAM fluorophore and 3' with a quencher. Each of the three LOCS reporters contains a different substrate for the nuclease (MNAzyme) in its loop region, but all three contain the same stem sequence (Stem 2).

[0746] Oligonucleotides

[0747] Oligonucleotides specific for this experiment include: forward primer 10 (SEQ ID NO:72), reverse primer 10 (SEQ ID NO:73), LOCS-2 (SEQ ID NO:2), LOCS-14 (SEQ ID NO:78), LOCS-15 (SEQ ID NO:79), partzyme A8 (SEQ ID NO:31), partzyme B8 (SEQ ID NO:32), partzyme A12 (SEQ ID NO:74), partzyme B12 (SEQ ID NO:75), partzyme A13 (SEQ ID NO:76), and partzyme B13 (SEQ ID NO:77). The sequences are listed in the sequence listing. The oligonucleotides specific for amplification of the TFRC gene are forward primer 10 (SEQ ID NO:72) and reverse primer 10 (SEQ ID NO:73). The oligonucleotides specific for detection of the TFRC amplicon and cleavage of LOCS-2 are partzyme A8 (SEQ ID NO:31) and partzyme B8 (SEQ ID NO:32). Oligonucleotides specific for detection of the TFRC amplicon and cleavage of LOCS-14 are partzyme A12 (SEQ ID NO: 74) and partzyme B12 (SEQ ID NO: 75). Oligonucleotides specific for detection of the TFRC amplicon and cleavage of LOCS-15 are partzyme A13 (SEQ ID NO: 76) and partzyme B13 (SEQ ID NO: 77).

[0748] Reaction conditions

[0749] use CFX96 thermal cycler performs real-time amplification and detection of target sequences in a total reaction volume of 20 μL. Cycling parameters are: 95°C for 2 minutes, 95°C for 5 seconds and 61°C for 30 seconds for 10 touchdown cycles (0.5°C decrement per cycle) and 95°C for 5 seconds and 52°C for 40 seconds for 35 cycles (data collected at 52°C steps). Melting curve parameters are 0.5°C increments from 20°C to 90°C for 5 seconds (keeping data acquisition). All reactions are repeated and contain 40nM of each forward primer, 200nM of each reverse primer, 200nM of each partenzyme A, 200nM of each partenzyme B, 200nM of each LOCS reporter, 2mM MgCl2 (Bioline) and 1x SensiFAST probe No-ROX mixture (Bioline). The reactions contained either a G-Block template (10,000 copies) homologous to the human transferrin receptor (TFRC) gene or no target (NF H2O).

[0750] result

[0751] In this experiment, PCR amplification, signal detection, and melt curve differentiation were performed in a single tube as a single-plex reaction. During PCR, three MNAzymes (MNAzyme 8, MNAzyme 12, and MNAzyme 13) were used to monitor the amplification of the target nucleic acid in real time by cleaving their corresponding LOCS reporters (LOCS-2, LOCS-14, and LOCS-15, respectively). All three MNAzymes were designed to detect the same target sequence (TFRC gene), however, each MNAzyme could hybridize and cleave a different substrate sequence (loop); thus, opening a different LOCS reporter.

[0752] Figure 14 The results shown show the corresponding melting curve characteristics obtained in the FAM channel after PCR amplification from reactions containing 10,000 copies of the TFRC gene target (black line) or without the target (grey line). The PCR amplification curves were crossed over the threshold (data not shown) and the Tm values ​​of 49°C, 48°C, and 49°C, respectively, associated with the cleavage of LOCS-2, LOCS-14, and LOCS-15. Figure 14 The presence of the TFRC gene was confirmed by the presence of melting peaks at 14A, 14B, and 14C). The absence of PCR amplification curves exceeding the threshold (data not shown) and the absence of cleavage peaks corresponding to uncleaved LOCS-2, LOCS-14, and LOCS-15 (respectively) were observed. Figure 14 The absence of the TFRC gene was confirmed by melting peaks at Tm of 74°C, 75°C, and 75°C associated with 14A, 14B, and 14C. Results are the average of replicate reactions plotted using Microsoft Excel (version 14). The data from this example demonstrates that the same universal stem region can be used with different substrates (loops) to produce comparable melting curve characteristics. Engineering LOCS reporters with different substrates using the same stem sequence facilitates the simple design of highly multiplexed assays and enables the rapid and easy adaptation of LOCS probes to detect any selected gene target.

[0753] Example 10 - Use of nicking endonucleases as an alternative method for opening LOCS reporters

[0754] In the following examples, a nicking endonuclease (Nt.AlwI) was used to demonstrate that comparable melting signals were generated when the loop portion of the LOCS reporter was opened after cleavage by a nicking endonuclease as an alternative to MNAzyme cleavage. Figure 3B illustrates the general strategy. In this example, a dual-labeled LOCS reporter contains a loop region that is complementary to the target sequence and a stem region that is non-complementary to the target sequence. Hybridization of the loop region of the LOCS reporter to the target completes the nicking endonuclease recognition site, thereby facilitating cleavage of the LOCS reporter by the nicking endonuclease and leaving the target intact. Because intramolecular bonds are stronger than intermolecular bonds, the stem region of the intact LOCS structure will melt at a higher temperature than the stem of the open, cleaved LOCS structure.

[0755] Oligonucleotides

[0756] Oligonucleotides specific for this experiment include: AF-NE-TV1 (SEQ ID NO: 82), LOCS-16 (SEQ ID NO: 80), AF-NE-R5b (SEQ ID NO: 83), and LOCS-17 (SEQ ID NO: 81). The sequences are listed in the sequence listing.

[0757] Reaction conditions

[0758] use The CFX96 thermal cycler performs real-time detection of target sequences in a total reaction volume of 20 μL. The reactants were incubated at a constant temperature of 52°C and data were collected every 20 seconds for a total of 200 data collections. The melting curve parameters were 0.5°C increments from 20°C to 90°C for 5 seconds (maintaining data acquisition). All reactions were performed in duplicate and contained 5mMMgCl2 (Ambion), 1x PCR buffer II (ABI), 4 units of Nt.AlwI (NEB) and 200nM LOCS (LOCS-16 or LOCS-17). Reactions contained AF-NE-TV1 or AF-NE-R5b (200nM) or no target (NF H2O).

[0759] result

[0760] Perform signal detection and melt curve differentiation in a single tube. Figure 15The results shown in A and 15B show the melting curve characteristics of LOCS-16 and LOCS-17 obtained in the FAM channel from reactions containing 200 nM of target (black line; AF-NE-TV1 or AF-NE-R5b, respectively) or no target (grey line; NF H2O). The results are the average of replicate reactions plotted using Microsoft Excel (version 14). The presence of target AF-NE-TV1 was detected by a rapid increase in fluorescence over time and the presence of a melting peak at 29°C corresponding to cleaved LOCS-16. The absence of target AF-NE-TV1 was determined by: (i) no increase in fluorescence over time, (ii) the absence of a melting peak at 29°C, and (iii) the presence of a melting peak at 65°C corresponding to uncleaved LOCS-16 ( Figure 15 A). The presence of target AF-NE-R5b was detected by a rapid increase in fluorescence over time and a melting peak at 48°C corresponding to cleaved LOCS-17. The absence of target AF-NE-R5b was determined by (i) no increase in fluorescence over time, (ii) the absence of a melting peak at 48°C, and (iii) the presence of a melting peak at 76°C corresponding to uncleaved LOCS-17 ( Figure 15 B).

[0761] The data from this example demonstrates that LOCS reporters can be used with alternative target detection methods, such as methods using nicking endonucleases. Furthermore, the data demonstrate that LOCS reporters containing the same stem (Stem 2) produce peaks at the same melting temperature (approximately 50°C), regardless of the loop degradation mechanism, such as cleavage by a nicking enzyme or an MNAzyme. In the current example, intact and open LOCS reporters containing Stem 2 produce melting peaks at 48°C and 76°C, respectively. These melting peaks are comparable to those produced using Stem 2 and an MNAzyme, such as Figure 4 D. Although small shifts of 1-2°C may be observed using LOCS containing the same stem in different protocols (e.g., ring cleavage by nicking enzymes versus ring cleavage by MNAzymes), these shifts may reflect differences in the reaction environment, where the concentration of salts, glycerol, or other components can affect the Tm of the stem.

[0762] Example 11 - Use of TaqMan Exonuclease as an Alternative Method for Opening LOCS Reporters

[0763] In the following examples, a TaqMan / hydrolysis probe-like approach is used to demonstrate that comparable melting characteristics are generated when the stem portion of a LOCS reporter is opened following exonuclease degradation of the loop as an alternative to MNAzyme cleavage. Figure 3A general strategy for this example is shown in Figure 2. In this example, a dual-labeled LOCS reporter contains a loop region that is complementary to the target sequence and a stem region that is not complementary to the target sequence. During PCR amplification, the loop region of the LOCS reporter can hybridize with the target and / or amplicon sequence. During the primer extension phase of PCR, the upstream primer extends into the region where the LOCS reporter hybridizes with the target, and the polymerase gradually cleaves the 5' end of the LOCS reporter loop region but keeps the stem region intact. The degradation of the loop region reduces the melting temperature of the LOCS stem because the intermolecular forces between the two LOCS stem fragments are significantly weaker than the intramolecular forces that occur therebetween within the closed LOCS molecule.

[0764] Oligonucleotides

[0765] Oligonucleotides specific for this experiment include: forward primer 2 (SEQ ID NO: 9), reverse primer 2 (SEQ ID NO: 10), and LOCS-18 (SEQ ID NO: 84). The sequences are listed in the sequence listing.

[0766] Reaction conditions

[0767] use CFX96 thermal cycler performs real-time amplification and detection of target sequences in a total reaction volume of 20 μL. Cycling parameters are: 95°C for 2 minutes, and 95°C for 5 seconds and 52°C for 40 seconds for 50 cycles (data are collected at 52°C steps). Melting curve parameters are 0.5°C increments from 20°C to 90°C for 5 seconds (keeping data acquisition). All reactions are repeated and contain 400nM of each primer, 200nM of LOCS-16, 2mM MgCl2 (Bioline) and 1x SensiFAST Probe No-ROX mixture (Bioline). Reactions contain synthetic G-Block templates (10,000 copies) or do not contain target (NF H2O).

[0768] result

[0769] In this experiment, PCR amplification, signal detection, and melting curve differentiation were performed in a single tube. The presence of the target gene (TV-btub) was detected by the presence of a PCR amplification curve and a melting peak at a Tm of 40°C corresponding to degraded open LOCS-18. The absence of the target gene was determined by the absence of a PCR amplification curve, a melting peak at a Tm of 40°C, and a melting peak at a Tm of 61°C corresponding to uncleaved closed LOCS-18. Figure 16The results shown in A and 16B show the corresponding amplification curves and melting curve characteristics obtained from reactions containing 10,000 copies of the gene target (black line) or no target (grey line) in the FAM channel. The results are the averages of replicate reactions plotted using Microsoft Excel (version 14).

[0770] The data from this example demonstrates that LOCS reporters can be used with alternative target detection methods that use a mechanism similar to that of TaqMan / hydrolysis probes, i.e., a polymerase with exonuclease activity degrades sequences that hybridize to the amplicon. Essentially, in this example, the LOCS probe consists of a loop portion that acts as a standard TaqMan probe sequence, but the "TaqMan-like probe sequence" has complementary stem sequences appended 5' and 3' to the hybridization region that are not themselves complementary to the target, but are complementary to each other. Furthermore, the data also demonstrate that LOCS reporters containing the same stem (Stem 1) produce peaks at the same melting temperature regardless of the mechanism of loop breakage, e.g., exonuclease degradation versus MNAzyme cleavage. In the current example, closed and open LOCS reporters containing Stem 1 produced melting peaks at 61°C and 40°C, respectively. These melting peaks are comparable to those produced using Stem 1 and MNAzymes, as shown in Figure 5. Figure 5 Compared with other exonuclease-based melting curve methods (such as TOCE), the advantages of the current method are that the released labeled probe fragments do not need to be subsequently hybridized with the capture probe to detect the presence of the target sequence, and because the fluorophore and quencher are locked in close proximity, the LOCS reporter can be better quenched initially.

[0771] Example 12: Method for simultaneous detection and quantification of multiple targets in a channel using two acquisition temperatures during amplification

[0772] The following example demonstrates a method in which a LOCS reporter allows for simultaneous detection and quantification of multiple targets in a single fluorescence channel by acquiring fluorescence readings at two discrete temperatures in real time during PCR. This strategy eliminates the need for post-PCR melt curve analysis, as demonstrated in the various examples above. Furthermore, this example describes two alternative methods for data analysis that facilitate quantification of the amount of any target present in a sample.

[0773] In this example, by heating the cells at two different temperatures (T L and T H), using two LOCS reporters comprising different stem lengths, compositions, and melting temperatures to simultaneously detect, differentiate, and quantify two targets, X and Y, in a single fluorescence channel. The assay was designed such that target X was monitored using LOCS-X (cleavable by MNAzyme X only in the presence of target X), and target Y was monitored using LOCS-Y (cleavable by MNAzyme Y only in the presence of target Y). Further, the assay was designed such that the Tm of open LOCS-X was lower than that of open LOCS-Y.

[0774] Select a lower detection temperature (T L ), causing the stem of the open LOCS-X to melt (dissociate), resulting in an increase in fluorescence, while the stem of the closed LOCS-X and the open and closed LOCS - The stem of Y will remain associated and quenched. Selecting a higher temperature (T H ), causing the stem of open LOCS-Y to melt (dissociate), resulting in increased fluorescence, while the stem of closed LOCS-Y remains associated and quenched. Since the stem of LOCS-X has a lower Tm, it will melt (dissociate) at this higher temperature, resulting in increased fluorescence regardless of whether it is in the open or closed state; however, the fluorescence produced by closed LOCS-X will only increase the background / baseline.

[0775] Can be used in T L and T H Two PCR amplification curves were drawn based on the fluorescence measurement results obtained at the same temperature. L Figure (threshold X; TX) and T H The graph (threshold Y; TY) sets the threshold for determining the presence of target X and / or Y. The thresholds (TX and TY) and various endpoints where the reaction is known to plateau can be predetermined based on previous experiments where the reaction containing only target X is at T L At the terminal EX1 or at T H The reaction containing only target Y reached a plateau at T H The reaction containing target X and target Y reaches a plateau at the endpoint EY1, and the reaction containing target X and target Y reaches a plateau at T H Optionally, endpoints EX1, EX2, EY1, and EY2 can be obtained from a positive control run in parallel with the experimental samples.

[0776] About T L If the PCR product exceeds TX and reaches a plateau at endpoint EX1, this result will indicate the presence of cleaved LOCS-X associated with target X. If target Y is also present, the amplification curve will not be affected because cleaved LOCS-Y is present at T LNo fluorescence will be produced below this level. Therefore, the Cq value obtained from the amplification curve exceeding TX allows quantification of target X in the sample.

[0777] Regarding T H amplification plot, if PCR generates an amplification curve that exceeds TY and reaches a plateau at EY1 or EY2, it indicates the presence of target Y. The threshold TY is set to be greater than the value EX2, so the amplification curve from a reaction containing only target X will not exceed this threshold. When both target X and Y are present, lysis of LOCS-X and LOCS-Y will produce an amplification curve that exceeds TY and reaches a plateau at EY2 greater than EY1, which is related to the lysis of LOCS-Y when only target Y is present. Since EX2 < TY < EY1 < EY2 and EX2 ≠ TY ≠ EY1 ≠ EY2, the endpoint where the amplification curve reaches a plateau can indicate the presence of target X, Y, or both. However, the Cq value obtained from the T H amplification curve exceeding TY will be affected by the amounts of target X and Y, and thus is only semi-quantitative for target Y. Various analytical methods (quantification methods 1 and 2) for adjusting the Cq value to accurately quantify the amount of target Y are described below.

[0778] Target Y quantification method 1

[0779] At T L 39 °C and T H 72 °C, the two amplification curves separately generated by the lysed LOCS-X species have the same efficiency and Cq, but reach a plateau at different endpoints EX1 and EX2 respectively. Therefore, the fluorescence signal (F-LOCS-X [[ID=ID=18]] FAF ) generated by the lysed LOCS-X species in the amplification curve at 72 °C can be inferred from the amplification curve at 39 °C by applying a fluorescence adjustment factor (FAF). FAF is the ratio of endpoints EX1 and EX2. The total fluorescence signal (F-total) in the amplification curve at 72 °C comes from the fluorescence signals generated by the lysed LOCS-X (F-LOCS-X FAF ) and LOCS-Y (F-LOCS-Y) species. Accordingly, the amplification curve generated by LOCS-Y at 72 °C can be inferred by the following method:

[0780] Because F-total = F-LOCS-X FAF + F-LOCS-Y,

[0781] F-LOCS-Y = F-toatal - F-LOCS-X FAF

[0782] ∴ The amplification curve at 72 °C generated by LOCS-Y

[0783] = The experimental amplification curve at 72 °C

[0784] Amplification curve of the experiment at -39℃*FAF

[0785] = Experimental amplification curve at 72℃

[0786]

[0787] Use TY from LOCS-Y in T H The Cq values ​​obtained from amplification curves generated at 72°C are fully quantitative for target Y. The formula can be applied regardless of whether the sample contains targets X, Y, or both to determine correct quantification.

[0788] Target Y Quantification Method 2

[0789] In the presence of both targets X and Y, the experimentally determined Cq value at 72°C (Cq 观察值 ) is shifted from the Cq value of a sample containing the same concentration of target Y but without target X. The Cq value determined from the standard curve of target Y at 72°C without target X is 观察值 The relationship between the expected Cq value (Cq 靶Y标准曲线 ) as follows:

[0790] Cq 观察值 =Cq 靶Y的标准曲线+ Relative offset in Cq

[0791] Among them, Cq 靶Y的标准曲线 =f(copy number of target Y)

[0792] and

[0793] Relative shift in Cq = f(copy number of target Y)

[0794] Because Cq 靶Y的标准曲线 The relative shift in Cq is a function of the copy number of target Y, so target Y can be determined based on the experimentally determined Cq value (Cq 观察值 ) is calculated. This relative shift in Cq values ​​has a mathematical relationship (logical relationship) with the natural logarithm of (copy number of target Y / copy number of target X). If the Cq value is determined using half of the maximum fluorescence level in the amplification curve measured at 72°C as the threshold, the logical relationship can be expressed as the following formula:

[0795]

[0796] Where y = relative shift in Cq, L = maximum possible shift in Cq, k = steepness factor, and x = ln (copy number of target Y / copy number of target X); note that the copy number of target X was determined from the amplification curve at 39°C.

[0797] Incorporating the offset factor in the Cq values ​​into the standard curve equation for target Y determined in the absence of target X enables correction of the Cq values ​​for the presence of target X and subsequently results in correct quantification of target Y.

[0798] The strategy described above can be adapted to detect any target, but in this particular example, target X is the CTcry gene and target Y is the NGopa gene; during PCR, the target is detected at low temperature (T L 39℃) and higher temperatures (T H The accumulation of amplicons for each target was monitored at 72°C; and both LOCS were labeled with the same fluorophore (JOE).

[0799] Oligonucleotides

[0800] Oligonucleotides specific for this experiment include: LOCS-19, LOCS-20, partzyme A9, partzyme B9, partzyme A10, partzyme B10, forward primer 7, reverse primer 7, forward primer 8, and reverse primer 11. The sequences are listed in the sequence listing. The oligonucleotides specific for CTcry amplification and quantification are LOCS-19, partzyme A9, partzyme B9, forward primer 7, and reverse primer 7. The oligonucleotides specific for NGopa amplification and quantification are LOCS-20, partzyme A10, partzyme B10, forward primer 8, and reverse primer 11.

[0801] Reaction conditions

[0802] use The CFX96 thermal cycler performs real-time detection of target sequences in a total reaction volume of 20 μL. The cycling parameters are: 95°C for 2 minutes, followed by 10 touchdown cycles of 95°C for 5 seconds and 61°C for 30 seconds (0.5°C decrement per cycle) and 40 cycles of 95°C for 5 seconds, 52°C for 40 seconds, 39°C for 1 second and 72°C for 1 second (data collected at 39°C and 72°C steps). Each reaction contained 40 nM of each forward primer, 200 nM of each reverse primer, 200 nM of each partial enzyme, 100 nM of LOCS-19 reporter, 200 nM of LOCS-20 reporter and 1xPlexMastermix (Bioline). The reactions did not contain target (NF H2O), synthetic G-Block X (20,000, 4,000, 800, 160, or 32 copies), synthetic G-Block of NGopa gene (20,000, 4,000, 800, 160, or 32 copies), various concentrations of CTcry gene in the background of synthetic G-Block of CTcry gene (20,000, 4,000, 800, 160, or 32 copies) or various concentrations of synthetic G-Blocks (20,000, 4,000, 800, 160 or 32 copies) of the NGopa gene in the context of synthetic G-Blocks (20,000, 4,000, 800, 160 or 32 copies) of the CTcry gene.

[0803] result

[0804] During PCR, two MNAzymes (MNAzyme 9 and MNAzyme 10) were used to monitor the amplification of the target nucleic acid in real time by cleaving their corresponding LOCS reporters (LOCS-19 and LOCS-20, respectively). MNAzyme 9 was designed to detect sequences homologous to the CTcry gene used to detect Chlamydia trachomatis and to cleave and open LOCS-19. MNAzyme 10 was designed to detect sequences homologous to the NGopa gene used to detect Neisseria gonorrhoeae and to cleave and open LOCS-20.

[0805] Figure 17The results shown in Figures A and 17B show comparative amplification curves obtained from reactions containing 20,000 copies of CTcry or NGopa or 20,000 copies of the gene target in the JOE channel, measured at 39°C and 72°C, respectively. The results are plotted as the average of replicate reactions. At 39°C, the sample containing 20,000 copies of the CTcry template (solid black line) shows an amplification curve in which the fluorescence level exceeds TX (black horizontal line denoted as TX) and reaches EX1 (black horizontal line denoted as EX1). At 39°C, the sample containing 20,000 copies of the CTcry and NGopa templates (solid gray line) shows an amplification curve in which the fluorescence level exceeds TX and reaches EX1, while the sample without the CTcry template (dashed black line) does not exceed TX and does not reach EX1. Therefore, an amplification curve that reaches a plateau at EX1 at 39°C confirms the presence of CTcry in the sample. The graph shows that samples containing 20,000 copies of CTcry alone (solid black line) and 20,000 copies of both CTcry and NGopa (solid grey line) have similar Cq values.

[0806] Since the Cq value at 39°C is not affected by the presence of NGopa, it can be used to quantify CTcry in a sample. Table 6 summarizes the quantification results of CTcry in samples with all combinations of 0, 32, 160, 800, 4,000, and 20,000 copies of CTcry and 0, 32, 160, 800, 4,000, and 20,000 copies of the NGopa gene target. The null value refers to the case where the Cq value was not determined at 39°C and therefore CTcry was not present in the sample.

[0807] Table 6: Determination of CTcry copy numbers in samples containing different copy numbers of CTcry and NGopa

[0808]

[0809] Figure 17B shows an amplification curve for a sample containing 20,000 copies of the NGopa template (dashed black line) at 72°C, where the fluorescence level exceeds TY (black horizontal line indicates TY) and reaches a plateau at EY1 (black horizontal line indicates EY1). At 72°C, a sample containing 20,000 copies of CTcry and NGopa (solid gray line) shows an amplification curve with a fluorescence level exceeding TY and reaching a plateau at EY2 (black horizontal line indicates EY2), while a sample without the target NGopa template (solid black line) does not exceed TY and does not reach EY1 or EY2, but only reaches a plateau at the endpoint EX2 (black horizontal line indicates endpoint EX2). Therefore, the amplification curve at 72°C reaching a plateau at EX2 confirms the presence of target CTcry but not NGopa in the sample; reaching a plateau at EY1 confirms the presence of NGopa but not CTcry in the sample; and reaching a plateau at EY2 confirms the presence of both CTcry and NGopa in the sample.

[0810] At 72°C, the Cq value of the sample containing 20,000 copies of CTcry and NGopa (solid gray line) is different from the Cq value of the sample containing only 20,000 copies of NGopa template (dashed black line). In this example, the presence of CTcry in the sample can shift the Cq value of NGopa by up to approximately -4.77 at 72°C. Therefore, assuming a PCR amplification efficiency of 100%, the Cq value at 72°C is used without normalization to determine the concentration of NGopa at 2 4.77 Table 7 summarizes the Cq values ​​for CTcry and Table 8 summarizes the amplification analysis of samples at 72°C for all combinations of 0, 32, 160, 800, 4,000, and 20,000 copies of the NGopa gene target without normalization. Null values ​​represent cases where no Cq value was determined at 72°C and therefore NGopa was not present in the sample. The results show that the method without normalization is fully quantitative for CTcry and semi-quantitative for NGopa.

[0811] Table 7: Cq values ​​of amplification curves of samples containing different copy numbers of CTcry and NGopa genes at 72°C without normalization

[0812]

[0813] Table 8: Determination of the copy number of the NGopa gene in samples containing different copy numbers of the CTcry and NGopa genes without normalization

[0814]

[0815] Figure 18 The results shown show comparative amplification curves obtained from reactions of all possible compositions containing 0, 32, and 20,000 copies of CTcry or 0, 32, and 20,000 copies of the NGopa gene target in the JOE channel, measured at 39°C and 72°C, respectively. The results are plotted as the average of replicate reactions. The samples represented in the figure contain the indicated amount of NGopa on the left side of the figure. The sample containing 20,000 copies of CTcry is represented by a black dashed line, the sample containing 32 copies of CTcry is represented by a gray solid line, and the sample containing 0 copies of CTcry is represented by a black solid line. Figure 18 The non-normalized amplification curves obtained at 72°C in B and 18E show that the presence of CTcry in the samples shifts the Cq values. The results were further analyzed using quantification method 1. The amplification curve at 39°C was subtracted from the amplification curve at 72°C by multiplying the FAF (because F-LOCS-Y = F-total - F-LOCS-X). FAF ) yielded a contribution corresponding to the cleaved LOCS-20 species but not the cleaved LOCS-19 ( Figure 18 All the inferred amplification curves at 72°C showed similar Cq values, in which the same amount of NGopa template was present, regardless of the amount of CTcry in the sample. Figure 18 I shows that in the case where the sample does not contain any NGopa template, the inferred amplification curve at 72°C does not show any significant amplification. Table 9 (F-LOCS-X FAF Normalized Cq values) and Table 10 (F-LOCS-X FAF The quantification of NGopa after normalization further demonstrated that F-LOCS-X FAF The effect of signal subtraction normalization is achieved by subtracting F-LOCS-X FAF Signals were normalized and analyzed for amplification of all combinations of samples with 0, 32, 160, 800, 4,000, and 20,000 copies of CTcry and 0, 32, 160, 800, 4,000, and 20,000 copies of the NGopa gene target at 72° C. Null values ​​are cases where no Cq value was determined at 72° C. and therefore target Y was not present in the sample.

[0816] Table 9: Cq values ​​of amplification curves at 72°C for samples containing different copy numbers of CTcry and NGopa genes after normalization with FAF

[0817]

[0818] Table 10: Determination of the copy number of NGopa in samples containing different copy numbers of CTcry and NGopa genes after normalization with FAF

[0819]

[0820]

[0821] For the amplification curves of the data obtained at 72° C., the Cq values ​​of the samples containing CTcry and NGopa were shifted relative to the samples containing only NGopa. The results were further analyzed using quantification method 2. Figure 19 Shown is the logarithmic regression of the natural logarithm of (copy number of NGopa / copy number of CTcry) plotted against the relative shift in Cq, where the Cq threshold is set to half the maximum fluorescence level in the amplification curve, according to the following formula:

[0822]

[0823] The above formula can be incorporated into the standard curve equation for NGopa determined in the absence of CTcry at 72°C as follows:

[0824]

[0825] Since the copy number of CTcry was determined based on data obtained at 39 °C, the above formula can be used to determine the experimental Cq (Cq) from 72 °C by a mathematical solvation procedure. 观察值 Table 11 shows the Cq values ​​observed at 72°C as the average of replicates, where the relative shift factor in Cq was calculated for samples with all combinations of 0, 32, 160, 800, 4,000, and 20,000 copies of the CTcry gene and 0, 32, 160, 800, 4,000, and 20,000 copies of the NGopa gene target. Table 12 shows the determination of copy number using Cq values ​​normalized by the relative shift factor for the same sample.

[0826] Table 11: Experimental Cq values ​​of amplification curves of samples containing different copy numbers of CTcry and NGopa and relative offset factors at 72°C

[0827]

[0828] Table 12: Determination of the copy number of NGopa in samples containing different copy numbers of CTcry and NGopa after normalization using the relative offset factor

[0829]

[0830] Example 13: Simultaneous detection of six targets in two fluorescent channels using six LOCS reporters

[0831] In the following examples, LOCS reporters are used to increase the number of targets that can be detected across two fluorescence channels. In this example, three targets are detected in each of the two fluorescence channels. In this example, all six LOCS reporters are labeled 5' with a fluorophore (JOE or Atto101) and 3' with a quencher (3IABkFQ or 3IAbRQSp, respectively). The loop region of the LOCS reporter each contains a different nucleic acid substrate, and the stem region contains a series of complementary base pairs that confine the LOCS reporter to a loop-stem configuration. In this configuration, the fluorophore and quencher are very close and quench fluorescence in the absence of a target.

[0832] Oligonucleotides

[0833] Oligonucleotides specific for this experiment include: LOCS-21 (SEQ ID NO:88), LOCS-22 (SEQ ID NO:89), LOCS-23 (SEQ ID NO:90), LOCS-24 (SEQ ID NO:91), LOCS-25 (SEQ ID NO:92), LOCS-26 (SEQ ID NO:93), partzyme A14 (SEQ ID NO:94), partzyme B14 (SEQ ID NO:95), partzyme A15 (SEQ ID NO:96), partzyme B15 (SEQ ID NO:97), partzyme A16 (SEQ ID NO:98), partzyme B16 (SEQ ID NO:99), partzyme A17 (SEQ ID NO:100), partzyme B17 (SEQ ID NO:101), partzyme A18 (SEQ ID NO:102), partzyme B18 (SEQ ID NO:103), partzyme A19 (SEQ ID NO:104), partzyme B19 (SEQ ID NO:105).

[0015] The oligonucleotides specifically for gpd amplification and detection are LOCS-21, partzyme A14, partzyme B14, forward primer 12, and reverse primer 12. The oligonucleotides specific for gpd3 amplification and detection are LOCS-22, partzyme A15, partzyme B15, forward primer 9, and reverse primer 12. The oligonucleotides specific for porA amplification and detection are LOCS-23, partzyme A16, partzyme B16, forward primer 13, and reverse primer 13. The oligonucleotides specific for TV-Btub amplification and detection are LOCS-24, partzyme A17, partzyme B17, forward primer 14, and reverse primer 2. The oligonucleotides specific for MgPa amplification and detection are LOCS-25, partzyme A18, partzyme B18, forward primer 1, and reverse primer 1.The oligonucleotides specific for LGV amplification and detection are LOCS-26, partzyme A19, partzyme B19, forward primer 15, and reverse primer 15.

[0834] Reaction conditions

[0835] use A CFX96 thermal cycler performs real-time amplification and detection of target sequences in a total reaction volume of 20 μL. Cycling parameters are: 40°C for 1 second, 70°C for 1 second, 85°C for 1 second, 95°C for 2 minutes, 10 touchdown cycles at 95°C for 5 seconds and 61°C for 30 seconds (0.5°C decrement per cycle), and 40 cycles at 95°C for 5 seconds, 52°C for 40 seconds, and 65°C for 1 second (data collected at the 65°C step), followed by 40°C for 1 second, 70°C for 1 second, and 85°C for 1 second. Melting curve parameters are 0.5°C increments from 20°C to 95°C, held for 5 seconds (holding data collection). All reactions were performed in duplicate 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 each of LOCS-21, LOCS-22, LOCS-24, and LOCS-25, and 150 nM each of LOCS-23 and LOCS-26, 8 mM MgCl2 (Bioline), 0.2 mM dNTPs (Bioline), 2 units of MyTaq polymerase (Bioline), and 1 x NH4 buffer (Bioline). The reactions contained G-Block templates (10,000 copies) homologous to gpd and / or gpd3 and / or porA or TV-Btub and / or MgPa and / or LGV genes, or no target (NF H2O).

[0836] result

[0837] Using an in vitro target amplification method known as PCR, six MNAzymes (MNAzyme 14, MNAzyme 15, MNAzyme 16, MNAzyme 17, MNAzyme 18, and MNAzyme 19) were used to monitor the amplification of target nucleic acids in real time by cleaving their corresponding LOCS reporters (LOCS-21, LOCS-22, LOCS-23, LOCS-24, LOCS-25, and LOCS-26, respectively). The presence of any or all targets in each channel (JOE and Texas Red) was determined by an increase in fluorescence during the PCR amplification phase (data not shown); individual targets were subsequently identified based on their unique LOCS melt signatures. MNAzyme 14 was designed to detect sequences homologous to the gpd gene and cleave and open LOCS-21; MNAzyme 15 was designed to detect sequences homologous to the gpd3 gene and cleave and open LOCS-22; MNAzyme 16 was designed to detect sequences homologous to the porA gene and cleave and open LOCS-23; MNAzyme 17 was designed to detect sequences homologous to the TV-Btub gene and cleave and open LOCS-24; MNAzyme 18 was designed to detect sequences homologous to the MgPa gene and cleave and open LOCS-25; and MNAzyme 19 was designed to detect sequences homologous to the LGV gene and cleave and open LOCS-26. In this experiment, amplification and detection were performed in a single tube containing all MNAzymes, primers, and LOCS oligonucleotides. The presence of gpd, gpd3, porA, TV-Btub, MgPa, LGV genes, or various combinations of these six genes (indicating samples with multiple infections) was detected by an increase in signal in one or both of the JOE and Texas Red channels (data not shown).

[0838] Figure 20-22 The results shown show the corresponding melting curve characteristics obtained after amplification from reactions containing target gpd ( Figure 20 A), gpd3( Figure 20 B), porA( Figure 20 C), gpd and gpd3 ( Figure 20 D), gpd and porA ( Figure 20 E), gpd3 and porA( Figure 20 F), or all three gpd, gpd3, and porA ( Figure 22 A) TV-Btub( Figure 21 A), MgPa( Figure 21 B)LGV( Figure 21 C), TV-Btub and MgPa ( Figure 21 D), TV-Btub and LGV ( Figure 21 E), MgPa and LGV ( Figure 21F), or 10,000 copies of all three TV-Btub, MgPa, and LGV (Figure 22B). Results are the average of replicate reactions plotted using Microsoft Excel (version 14).

[0839] The data from this example, summarized in Table 13, demonstrate that the LOCS melting profile generated in the presence of the gpd gene target (Tm = 53°C, 68°C, and 85°C) is different from the LOCS melting profile generated in the presence of the gpd3 (Tm = 30°C, 43°C, 77°C, and 85°C) and porA (Tm = 68°C and 77°C) gene targets. This example demonstrates that in addition to detecting gpd, gpd3, and / or porA, TV-Btub, MgPa, and / or LGV gene targets can be simultaneously detected in a second channel. The LOCS melting profile generated in the presence of the TV-Btub gene target (Tm = 30°C, 42°C, 66°C, and 82°C) is different from the LOCS melting profile generated in the presence of the MgPa (53°C, 66°C, and 82°C) and LGV (Tm = 68°C) gene targets. Further, in the presence of two or more gene targets read from a single channel, the LOCS melting profile is also different from the above-described melting profile in the presence of only a single gene target. In the JOE channel, when gpd and gpd3 gene targets were present in a single reaction, unique LOCS melting characteristics (Tm = 30°C, 43°C, 53°C, and 85°C) indicated that both targets were detected. Similarly, when gpd and porA or gpd3 and porA were present in a single reaction, unique melting curve characteristics (Tm = 53°C and 68°C and Tm = 30°C, 43°C, 68°C, and 77°C, respectively) specifically indicated which two targets were detected. Similarly, in the Texas Red channel, LOCS melting characteristics were unique for each combination of the following targets: TV-Btub and MgPa (Tm = 30°C, 42°C, 53°C, and 82°C), TV-Btub and LGV (Tm = 30°C, 42°C, and 68°C), or MgPa and LGV (Tm = 53°C and 68°C). When gpd, gpd3, and porA gene targets were all present in a single reaction, unique LOCS melting signatures (Tm = 30°C, 43°C, 53°C, and 68°C) in the JOE channel indicated that all three targets were detected. Similarly, in the Texas Red channel, the presence of a single TV-Btub, MgPa, and LGV target produced unique LOCS melting signatures (Tm = 30, 42°C, 53°C, and 68°C).

[0840] Table 13: Figure 20 、 21 Summary of the melting temperatures (Tm) of LOCS reporters in the presence of one, two, or three targets as shown in Figures 2 and 22.

[0841]

[0842] This example demonstrates that six targets co-amplified in a single well using two fluorescence channels (three targets detected in each channel) can be distinguished based on a unique LOCS melting signature. The example provides a simple method for detecting multiple targets in a single well using only two fluorescence channels. Due to different stem lengths and base pair compositions, the different stems (stem 5, stem 6, stem 7, stem 8, stem 9, and stem 10) within LOCS-21, LOCS-22, LOCS-23, LOCS-24, LOCS-25, and LOCS-26 produce unique fluorescent melting curve signatures.

[0843] Example 14: Simultaneous detection and discrimination of ten targets in a single well using five fluorescent channels and ten LOCS reporters

[0844] In the following examples, LOCS reporters are used to increase the number of targets that can be detected simultaneously in a single well using five fluorescent channels by increasing the number of targets that can be detected in each channel. In this example, two targets are detected in each of the five fluorescent channels. In this example, all ten LOCS reporters are labeled with fluorophores (FAM, JOE, AttoRho101, Cy5, or Cy5.5) at 5' and with quenchers (3IABkFQ or 3IAbRQSp) at 3'. The loop region of the LOCS reporter contains a nucleic acid substrate, and the stem region contains a series of complementary base pairs that confine the LOCS reporter to a loop-stem configuration. In this configuration, the fluorophore and quencher are very close and quench fluorescence in the absence of a target.

[0845] Oligonucleotides

[0846] Oligonucleotides specific for this experiment include: LOCS-10 (SEQ ID NO:40), LOCS-15 (SEQ ID NO:79), LOCS-21 (SEQ ID NO:88), LOCS-22 (SEQ ID NO:89), LOCS-24 (SEQ ID NO:91), LOCS-27 (SEQ ID NO:113), LOCS-28 (SEQ ID NO:114), LOCS-29 (SEQ ID NO:115), LOCS-30 (SEQ ID NO:116), LOCS-31 (SEQ ID NO:117), partzyme A9 (SEQ ID NO:43), partzyme B9 (SEQ ID NO:44), partzyme A10 (SEQ ID NO:48), partzyme B10 (SEQ ID NO:49), partzyme A14 (SEQ ID NO:94), partzyme B14 (SEQ ID NO:95), partzyme A15 (SEQ ID NO:97), partzyme B15 (SEQ ID NO:98), partzyme B16 (SEQ ID NO:99), partzyme B17 (SEQ ID NO:100), partzyme B18 (SEQ ID NO:101), partzyme B19 (SEQ ID NO:102), partzyme B110 (SEQ ID NO:103), partzyme B111 (SEQ ID NO:104), partzyme B112 (SEQ ID NO:105), partzyme B113 (SEQ ID NO:106), partzyme B114 (SEQ ID NO:107), partzyme B115 (SEQ ID NO:108), partzyme B116 (SEQ ID NO:109), partzyme B117 (SEQ ID NO:118), partzyme B118 (SEQ ID NO:119), partzyme NO:98), partzyme A16 (SEQ ID NO:98), partzyme B16 (SEQ ID NO:99), partzyme A17 (SEQ ID NO:100), partzyme B17 (SEQ ID NO:101), partzyme A18 (SEQ ID NO:102), partzyme B18 (SEQ ID NO:103), partzyme A19 (SEQ ID NO:104), partzyme B19 (SEQ ID NO:105), partzyme A20 (SEQ ID NO:118), partzyme B20 (SEQ ID NO:119), partzyme A21 (SEQ ID NO:120), partzyme B21 (SEQ ID NO:121), forward primer 1 (SEQ ID NO:7), reverse primer 1 (SEQ ID NO:8), reverse primer 2 (SEQ ID NO:10), forward primer 7 (SEQ ID NO:41), reverse primer 7 (SEQ ID NO:42), forward primer 8 (SEQ ID NO: NO:52), forward primer 9 (SEQ ID NO:54), reverse primer 9 (SEQ ID NO:55), forward primer 10 (SEQ ID NO:72), reverse primer 10 (SEQ ID NO:73), reverse primer 11 (SEQ ID NO:85), forward primer 12 (SEQ ID NO:106), reverse primer 12 (SEQ ID NO:107), forward primer 13 (SEQ ID NO:108), reverse primer 13 (SEQ ID NO:109), forward primer 14 (SEQ ID NO:110), reverse primer 14 (SEQ ID NO:111), forward primer 15 (SEQ ID NO:112), reverse primer 15 (SEQ ID NO:113), forward primer 15 (SEQ ID NO:114), reverse primer 15 (SEQ ID NO:115), forward primer 15 (SEQ ID NO:116), reverse primer 15 (SEQ ID NO:117),NO: 110), forward primer 15 (SEQ ID NO: 111), reverse primer 15 (SEQ ID NO: 112), forward primer 16 (SEQ ID NO: 122), and reverse primer 16 (SEQ ID NO: 123). The sequences are listed in the sequence listing.

[0847] The oligonucleotides specific for amplifying and detecting CTcry are LOCS-29, partzyme A9, partzyme B9, forward primer 7, and reverse primer 7. The oligonucleotides specific for amplifying and detecting NGopa are LOCS-15, partzyme A10, partzyme B10, forward primer 8, and reverse primer 11. The oligonucleotides specific for amplifying and detecting gpd are LOCS-21, partzyme A14, partzyme B14, forward primer 12, and reverse primer 12. The oligonucleotides specific for amplifying and detecting gpd3 are LOCS-22, partzyme A15, partzyme B15, forward primer 9, and reverse primer 9. The oligonucleotides specific for amplifying and detecting porA are LOCS-27, partzyme A16, partzyme B16, forward primer 13, and reverse primer 13. The oligonucleotides specific for TV-Btub amplification and detection are LOCS-24, partzyme A17, partzyme B17, forward primer 14, and reverse primer 2. The oligonucleotides specific for MgPa amplification and detection are LOCS-28, partzyme A18, partzyme B18, forward primer 1, and reverse primer 1. The oligonucleotides specific for LGV amplification and detection are LOCS-10, partzyme A19, partzyme B19, forward primer 15, and reverse primer 15. The oligonucleotides specific for polA amplification and detection are LOCS-30, partzyme A20, partzyme B20, forward primer 16, and reverse primer 16. The oligonucleotides specific for TFRC amplification and detection are LOCS-31, partzyme A21, partzyme B21, forward primer 10, and reverse primer 10.

[0848] Reaction conditions

[0849] use A CFX96 thermal cycler performs real-time amplification and detection of target sequences in a total reaction volume of 20 μL. Cycling parameters are: 31°C for 1 second, 42°C for 1 second, 53°C for 1 second, 60°C for 1 second, 95°C for 2 minutes, 10 touchdown cycles of 95°C for 5 seconds and 61°C for 30 seconds (0.5°C decrement per cycle), and 40 cycles of 95°C for 5 seconds, 52°C for 40 seconds, and 65°C for 5 seconds (data collected at the 65°C step), followed by 31°C for 1 second, 42°C for 1 second, 53°C for 1 second, and 60°C for 1 second. Melting curve parameters are 0.5°C increments from 20°C to 95°C, held for 5 seconds (holding data collection). All reactions were performed in duplicate 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 each of LOCS-10, LOCS-15, LOCS-21, LOCS-22, LOCS-24, LOCS-27, LOCS-29, and LOCS-31 reporters, and 150 nM each of LOCS-28 and LOCS-30, 8 mM MgCl2 (Bioline), 0.2 mM dNTPs (Bioline), 2 units of MyTaq polymerase (Bioline), and 1x NH4 buffer (Bioline). The reactions contained G-Block templates (10,000 copies) homologous to NGopa and / or porA, gpd and / or gpd3, TV-Btub and / or MgPa, CTcry and / or LGV, or polA genes, or no target (NF HO). All reactions contained a background of 10,000 copies of human genomic DNA containing the TFRC gene target as an endogenous control. Detection of the TFRC gene was monitored in each reaction by an increase in fluorescence in the Cy5.5 channel as an internal control (data not shown).

[0850] result

[0851] Using an in vitro target amplification method known as PCR, ten MNAzymes (MNAzyme 9, MNAzyme 10, and MNAzymes s14-21) were used to monitor the amplification of target nucleic acids in real time by cleaving their corresponding LOCS reporters (LOCS-29, LOCS-15, LOCS-21, LOCS-22, LOCS-27, LOCS-24, LOCS-28, LOCS-10, LOCS-30, and LOCS-31, respectively). The presence of one or two targets within each channel (FAM, HEX, Texas Red, Cy5, and Cy5.5) was determined by increasing fluorescence during the PCR amplification phase, followed by identification of individual targets using the LOCS melt signature.

[0852] MNAzyme 10 was designed to detect sequences homologous to the NGopa gene and cleave and open LOCS-15; MNAzyme 16 was designed to detect sequences homologous to the porA gene and cleave and open LOCS-27; MNAzyme 14 was designed to detect sequences homologous to the gpd gene and cleave and open LOCS-21; MNAzyme 15 was designed to detect sequences homologous to the gpd3 gene and cleave and open LOCS-22; MNAzyme 17 was designed to detect sequences homologous to the TV-Btub gene and cleave and open LOCS-24; MNAzyme 18 was designed to detect sequences homologous to the MgPa gene and cleave and open LOCS-28; MNAzyme 9 was designed to detect sequences homologous to the CTcry gene and cleave and open LOCS-29; and MNAzyme 19 was designed to detect sequences homologous to the LGV gene. MNAzyme 20 is designed to detect sequences homologous to the pmpH gene and cleave and open LOCS-10, MNAzyme 20 is designed to detect sequences homologous to the polA gene and cleave and open LOCS-30, and MNAzyme 21 is designed to detect the human TFRC gene and cleave and open LOCS-31.

[0853] In this experiment, amplification and detection were performed in a single tube containing all partzymes, all primers, and all LOCS oligonucleotides for all MNAzymes. The presence of any one of NGopa, porA, gpd, gpd3, TV-Btub, MgPa, CTcry, LGV, polA, TFRC, or various combinations of these ten gene targets (representing samples with multiple infections) was detected by an increase in signal in the FAM, HEX, Texas Red, Cy5, or Cy5.5 channels (data not shown).

[0854] The results shown in Figure 23 show the corresponding melting curve characteristics obtained from reactions containing 10,000 copies of the target NGopa (Figure 23A), porA (Figure 23B), NGopa and porA (Figure 23C), gpd (Figure 23D), gpd3 (Figure 23E), gpd and gpd3 (Figure 23F), TV-Btub (Figure 23G), MgPa (Figure 23H), TV-Btub and MgPa (Figure 23I), CTcry (Figure 23J), LGV (Figure 23K), CTcry and LGV (Figure 23L), polA (Figure 23M), or TFRC (Figure 23N) after amplification. The results are the average of replicate reactions plotted using Microsoft Excel (Version 14).

[0855] The data of this example summarized in Table 14 demonstrate that the LOCS melting characteristics produced in the presence of a single target are different from the LOCS melting characteristics produced in the presence of a second target that fluoresces in the same channel. In addition, the LOCS melting characteristics produced in the presence of two targets in a single channel are different from the LOCS melting characteristics produced by either of the two single targets detected at a specific wavelength. In this example, this result is consistent across all five channels. In the FAM channel, the LOCS melting characteristics produced in the presence of the NGopa (Tm = 53°C), porA (Tm = 30°C and 76°C), and NGopa and porA (Tm = 30°C and 53°C) gene targets are all different. In the HEX channel, the LOCS melting characteristics produced in the presence of the gpd (53°C and 70°C), gpd3 (30°C, 43°C, and 76°C), and gpd and gpd3 (30°C, 43°C, and 53°C) gene targets are all different. In the Texas Red channel, the LOCS melting profiles generated in the presence of the TV-Btub (31°C, 41°C, 59°C, and 83°C), MgPa (63°C), and TV-Btub and MgPa (31°C, 41°C, and 63°C) gene targets were all distinct. In the Cy5 channel, the LOCS melting profiles generated in the presence of the CTcry (61°C and 79°C), LGV (47°C and 80°C), and CTcry and LGV (47°C and 61°C) gene targets were all distinct. In this example, human genomic DNA (TFRC) was detected in the Cy5.5 channel in all reactions, thus serving as an endogenous control. Therefore, reactions detecting polA also detected TFRC, as evidenced by two peaks in the LOCS melting profiles corresponding to the TFRC and polA gene targets, respectively (39°C and 59°C). In the Cy5.5 channel, the LOCS melting signatures generated in the presence of polA and TFRC (39°C and 59°C) differ from those generated in the presence of TFRC alone (39°C and 80°C), thereby distinguishing reactions containing polA (polA and TFRC melting signatures) from reactions lacking polA (TFRC-only LOCS melting signatures).

[0856] Table 14: Summary of melting temperatures (Tm) of LOCS reporters in the presence of one or two targets as shown in Figure 23.

[0857]

[0858] This example demonstrates that ten gene targets co-amplified in a single well and using five fluorescence channels (two targets detected per well) can be distinguished based on unique LOCS melting signatures. The example provides a simple method for detecting multiple targets in a single well using five fluorescence channels, thereby doubling the multiplexing capacity of a single PCR machine.

[0859] Example 15: Method for single-channel, single-well multiplexing by measuring fluorescence at defined temperature points

[0860] The following example provides an alternative for detecting and discriminating multiple targets at a single wavelength without performing a full melting curve analysis over a wide temperature range, as illustrated previously in Examples 1 to 6. In this example, a method for detecting and identifying three targets at a single wavelength is demonstrated by using a modified LOCS protocol plus a selection of multiple analysis strategies. Three LOCS reporters X, Y, and Z can be used to detect targets X, Y, and Z, all of which can be labeled with the same fluorophore. Each of these three LOCS reporters includes a different stem region with a different melting temperature, where Tm of LOCS-X < Tm of LOCS-Y < Tm of LOCS-Z. Additionally, each of the LOCS reporters contains a different loop region that includes a substrate specific for MNA enzymes X, Y, or Z. MNA enzymes X, Y, or Z are designed to recognize and detect targets X, Y, or Z, respectively, and cleave LOCS reporters X, Y, or Z, respectively. A limited number of fluorescence measurements (six or fewer in total) can be obtained before and / or after PCR amplification of the targets. Various analysis methods can utilize the measurements taken at specific temperature and time points. Table 15 summarizes these measurements.

[0861] Table 15: Summary of analysis protocols allowing specific detection of three cleaved LOCS in a single channel using measurements of relative fluorescence units (RFU) collected at specific temperature points

[0862]

[0863] [[ID=!17]]

[0864] The strategy outlined in this example can be adapted to detect any target, but in this particular example, target X is the TFRC gene, target Y is the rpoB gene, and target Z is the pss gene, which are detected by probes LOCS-X (LOCS-22), LOCS-Y (LOCS-21), and LOCS-Z (LOCS-23), respectively, labeled with the same fluorophore (JOE). The various detection temperatures used are T Xa = 40.5 °C; T Xb = 56.5 °C; T Xc=78.5℃; T 0b =32℃; T Xb =40.5℃; T Yb =56.5℃; T Zb =65.5℃; T Xc =39.5℃; T Yc =56.5℃; T Zc =65.5℃; T Xd =40℃; T Yd =51°C; and T Zd = 65° C. Further, in the following analysis, a value of N = 0.5° C. is specified.

[0865] Analysis Plan A1

[0866] At the first specific temperature T Xa At 1 , cleavage of LOCS-X generates a significant fluorescence signal relative to the no-template control, and this will exceed the threshold FXa. At the same temperature, the presence of cleaved LOCS-Y and / or LOCS-Z does not contribute to the generation of a significant fluorescence signal due to the higher Tm of the stems of these LOCS, and therefore, the fluorescence level remains below the threshold FXa relative to the no-template control. Temperature T Xa Post-amplification measurement of fluorescence allows specific detection of cleaved LOCS-X.

[0867] At temperatures above T Xa The second specific temperature T Ya At temperature T, the cleaved LOCS-Y produces a relative fluorescence signal significantly greater than the threshold FYa. Ya At this temperature, the relative fluorescence signal generated by the cleaved LOCS-X and / or LOCS-Z will be significantly less than the threshold FYa. Ya Post-amplification measurement of fluorescence under RT allows specific detection of cleaved LOCS-Y.

[0868] At temperatures above T Xa and T Ya The third specific temperature T Za At temperature T, the cleaved LOCS-Z generates a relative fluorescence signal significantly greater than the threshold FZa. Za At this temperature, the relative fluorescence signal generated by the cleaved LOCS-X and / or LOCS-Y is significantly less than the threshold FZa. Za Post-amplification measurement of fluorescence under ΔΨ allows specific detection of cleaved LOCS-Z. Detection of each cleaved LOCS reporter indicates the presence of the corresponding target in the sample.

[0869] Analytical Plan A2

[0870] When one or more LOCS species are cleaved in a sample, an algorithm can be used to determine the cleaved LOCS species instead of using a threshold. This algorithm uses the following parameters at three detection temperatures (T Xa 、T Ya and T Za ) relative to the no-template control at each of the three detection temperatures. The ratio of the relative fluorescence levels measured at the three temperatures is unique for each of the possible combinations of cleaved LOCS species and serves as a fingerprint indicating the presence of one or more corresponding target genes in the sample. The algorithm includes a series of seven logical tests; one logical test is assigned to each possible combination of cleaved and uncleaved LOCS to determine the presence of one or more cleaved LOCS species. The possible combinations are (only X), (only Y), (only Z), (only X and Y), (only X and Z), (only Y and Z), or (X, Y, and Z). For a sample to be determined to be positive for a particular target combination, it needs to be determined to be true for a particular logical test. The logical tests determine (1) whether the ratio of the fluorescence signals measured at the three different temperatures is within a predefined range of values, and (2) whether the fluorescence signal is signi...

Claims

1. Use of a first closed stem-loop oligonucleotide and a second closed stem-loop oligonucleotide and an enzyme in the preparation of a diagnostic agent for a method for determining the presence or absence of a first target and a second target in a sample, wherein the enzyme is capable of cleaving or degrading the single-stranded loop portion of the first closed stem-loop oligonucleotide and the single-stranded loop portion of the second closed stem-loop oligonucleotide only when in contact with the target or an amplicon thereof, the method comprising: (a) preparing a reaction mixture by contacting a sample presumed to include the first target and / or the second target, or amplicons thereof, with (i) and (ii): (i) said first closed stem-loop oligonucleotide and said second closed stem-loop oligonucleotide, wherein each of said closed stem-loop oligonucleotides comprises a double-stranded stem portion of hybridized nucleotides linked to a closed single-stranded loop portion of unhybridized nucleotides, wherein: The first closed stem-loop oligonucleotide and the second closed stem-loop oligonucleotide differ in the number of hybridizing nucleotides and / or the sequence of hybridizing nucleotides in the stem portion, The closed single-stranded loop portions of the first closed stem-loop oligonucleotide and the second closed stem-loop oligonucleotide are capable of hybridizing to complementary sequences in the presence of the target or an amplicon thereof, and (ii) the enzyme, wherein the enzyme is selected from a catalytic nucleic acid, an exonuclease, or an endonuclease; (b) treating the reaction mixture: - under conditions suitable for said enzyme to induce cleavage or degradation of said loop portions of said first closed stem-loop oligonucleotide and said second closed stem-loop oligonucleotide to thereby produce a first open stem-loop oligonucleotide and a second open stem-loop oligonucleotide; - at a first temperature, at or above which the strands of the double-stranded stem portion of the first open stem-loop oligonucleotide dissociate to thereby promote spatial separation of the fluorophore molecule and the quencher molecule of the stem portion of the first open stem-loop oligonucleotide and provide a first detectable fluorescent signal, and - at a second temperature, at or above which the strands of the double-stranded stem portion of the second open stem-loop oligonucleotide dissociate to thereby facilitate spatial separation of the fluorophore molecule and the quencher molecule of the stem portion of the second open stem-loop oligonucleotide and provide a second detectable fluorescent signal; in: The first temperature is lower than the second temperature, and The fluorophore of the first open stem-loop oligonucleotide and the fluorophore of the second open stem-loop oligonucleotide emit in the same color region of the visible spectrum; and (c) detecting the level of the first detectable fluorescent signal and the second detectable fluorescent signal at one or more temperatures consisting of or equal to or greater than the second temperature to thereby determine the presence or absence of the target in the sample.

2. The use of claim 1 , wherein treating the reaction mixture under conditions suitable for the enzyme to induce the cleavage comprises an enzyme selected from the group consisting of an MNAzyme, a DNA enzyme, a ribozyme, an aptazyme, a restriction endonuclease, a nicking endonuclease, or a polymerase with exonuclease activity.

3. The use of claim 1 , wherein the enzyme comprises an MNAzyme, and the treating of the reaction mixture comprises treating the reaction mixture under conditions suitable for: A first MNAzyme binds to the first target or an amplicon thereof and the substrate arm of the first MNAzyme hybridizes to the loop portion of the first closed stem-loop oligonucleotide to thereby facilitate said cleavage of the loop portion of the first closed stem-loop oligonucleotide by the first MNAzyme to form the first open stem-loop oligonucleotide.

4. The use according to claim 3, wherein the first target is a nucleic acid sequence or an amplicon thereof capable of hybridizing to a sensor arm of the first MNAzyme to thereby facilitate assembly of the first MNAzyme.

5. The use according to claim 1, wherein: - the first target is an analyte; - the enzyme comprises an enzyme having an aptamer, the aptamer being capable of binding to the first target; and - The binding of the first target to the aptamer enables the enzyme having the aptamer to exhibit catalytic activity. The use according to claim 5 , wherein the enzyme having an aptamer comprises any one or more of the following: an apta-DNA enzyme, an apta-ribozyme, an apta-MNAzyme.

7. Use according to claim 5, wherein the analyte is a molecule.

8. The use according to claim 5, wherein the analyte is a protein or a compound.

9. The use according to claim 3, wherein: - the first target is an analyte; - the reaction mixture further comprises an oligonucleotide sequence capable of hybridizing to the sensor arm of the first MNAzyme to thereby facilitate assembly of the first MNAzyme; - said first MNAzyme comprises an aptamer sequence capable of binding to said first target; and - Binding of the first target to the aptamer sequence is capable of rendering the first MNAzyme catalytically active.

10. Use according to claim 9, wherein the analyte is a molecule.

11. The use according to claim 9, wherein the analyte is a protein or a compound.

12. Use according to any one of claims 1 to 11, wherein during said cleavage or degradation by said enzyme, said closed stem-loop oligonucleotide does not hybridize to said target or an amplicon thereof.

13. The method of claim 1 or 4, wherein the enzyme comprises a restriction endonuclease and the treatment of the reaction mixture comprises: The reaction mixture is treated under conditions suitable for hybridization of the first target or an amplicon thereof to the loop portion of the first closed stem-loop oligonucleotide to form a double-stranded sequence for association with a first restriction endonuclease and thereby promote the cleavage of the loop portion of the first closed stem-loop oligonucleotide to form the first open stem-loop oligonucleotide.

14. The use according to claim 13, wherein the restriction endonuclease is a nicking endonuclease capable of associating with and cleaving the loop chain of the double-stranded sequence for the first restriction endonuclease.

15. The use according to claim 1 or 4, wherein the enzyme has exonuclease activity and the treatment of the reaction mixture comprises: The reaction mixture is treated under conditions suitable for: - a first target or an amplicon thereof hybridizes to said loop portion of said first closed stem-loop oligonucleotide to form a first double-stranded sequence comprising said first target or an amplicon thereof, - a first primer oligonucleotide hybridizing to the first target or an amplicon thereof to form a second double-stranded sequence, the second double-stranded sequence being located upstream (5') relative to the first double-stranded sequence including the first target or an amplicon thereof - a first enzyme having exonuclease activity associates with said loop portion of said first closed stem-loop oligonucleotide at or near an end of said first primer oligonucleotide, and - catalytic activity of said first enzyme having exonuclease activity, to thereby promote degradation of the loop portion of said first double-stranded sequence comprising said first target or amplicon and to form said first open stem-loop oligonucleotide.

16. The method according to claim 1 or 4, wherein the enzyme has exonuclease activity and the treatment of the reaction mixture comprises: The reaction mixture is treated under conditions suitable for: - a first target or an amplicon thereof hybridizes to said loop portion of said first closed stem-loop oligonucleotide to form a first double-stranded sequence comprising said first target or an amplicon thereof, - a first enzyme having exonuclease activity associates with said first double-stranded sequence comprising said first target or an amplicon thereof, and - catalytic activity of said first enzyme having exonuclease activity, to thereby promote degradation of the loop portion of said first double-stranded sequence comprising said first target or amplicon and to form said first open stem-loop oligonucleotide.

17. The use according to claim 1, wherein the enzyme comprises a DNA enzyme and / or a ribozyme that requires a first cofactor for catalytic activity, and the treating of the reaction mixture comprises treating the reaction mixture under conditions suitable for: - the first cofactor binds to the DNA enzyme and / or the first cofactor binds to the ribozyme so that the DNA enzyme and / or the ribozyme exhibit catalytic activity, - a DNA enzyme and / or a ribozyme hybridizing to the loop portion of the first closed stem-loop oligonucleotide, - the catalytic activity of said DNA enzyme and / or said ribozyme, to thereby promote the cleavage of said loop portion of said first closed stem-loop oligonucleotide and form said first open stem-loop oligonucleotide, in: The first target is the first cofactor.

18. Use according to claim 17, wherein the first cofactor is a metal ion.

19. The use according to any one of claims 1 to 11, wherein said treating further comprises treating said reaction mixture under conditions suitable for any one or more of the following: - a second MNAzyme binds to the second target or amplicon thereof and the substrate arm of the second MNAzyme hybridizes to the loop portion of the second closed stem-loop oligonucleotide to thereby facilitate said cleavage of the loop portion of the second closed stem-loop oligonucleotide by the second MNAzyme to form the second open stem-loop oligonucleotide; - hybridizing a second target or an amplicon thereof to said loop portion of said second closed stem-loop oligonucleotide to form a double-stranded sequence for a second restriction endonuclease to associate with said double-stranded sequence and thereby promote said cleavage of said loop portion of said second closed stem-loop oligonucleotide, thereby forming said second open stem-loop oligonucleotide; - a second target or an amplicon thereof hybridizes to said loop portion of said second closed stem-loop oligonucleotide to form a second double-stranded sequence comprising said second target or an amplicon thereof, a second primer oligonucleotide hybridizing to the second target or an amplicon thereof to form a second double-stranded sequence, the second double-stranded sequence being located upstream (5') relative to the second double-stranded sequence including the second target or an amplicon thereof, a second enzyme having exonuclease activity associates with the loop portion of the second closed stem-loop oligonucleotide at or near an end of the second primer oligonucleotide, and catalytic activity of the second enzyme having exonuclease activity to thereby promote degradation of the loop portion of the second double-stranded sequence comprising the second target or amplicon and form the second open stem-loop oligonucleotide; - a second target or an amplicon thereof hybridizes to said loop portion of said second closed stem-loop oligonucleotide to form a second double-stranded sequence comprising said second target or an amplicon thereof, a second enzyme having exonuclease activity associates with the second double-stranded sequence comprising the second target or an amplicon thereof, and catalytic activity of the second enzyme having exonuclease activity to thereby promote degradation of the loop portion of the second double-stranded sequence comprising the second target or amplicon and form the second open stem-loop oligonucleotide; in: The second target is a nucleic acid.

20. The use according to any one of claims 1 to 11, wherein the treatment further comprises treating the reaction mixture under conditions suitable for: - the second cofactor binds to the DNA enzyme and / or the second cofactor binds to the ribozyme so that the DNA enzyme and / or the ribozyme exhibit catalytic activity, The DNA enzyme and / or ribozyme hybridizes to the loop portion of the second closed stem-loop oligonucleotide, the catalytic activity of the DNA enzyme and / or the ribozyme, thereby promoting the cleavage of the loop portion of the second closed stem-loop oligonucleotide and forming the second open stem-loop oligonucleotide, in: The second target is the second cofactor.

21. The use according to claim 20, wherein the second cofactor is a metal ion.

22. The use according to claim 19, wherein the second restriction endonuclease is a nicking endonuclease capable of associating with and cleaving the circular strand of the double-stranded sequence comprising the second target or an amplicon thereof.

23. The use of claim 19, wherein the nucleic acid sequence is capable of hybridizing to the sensor arm of the second MNAzyme to thereby facilitate assembly of the second MNAzyme.

24. The use according to any one of claims 1 to 11, wherein: - the second target is an analyte; - the enzyme comprises an enzyme having an aptamer, the aptamer being capable of binding to the second target; and - The binding of the second target to the aptamer can cause the enzyme having the aptamer to exhibit catalytic activity.

25. Use according to claim 24, wherein the analyte is a molecule.

26. The use according to claim 24, wherein the analyte is a protein or a compound.

27. The use according to claim 24, wherein the enzyme having an aptamer comprises any one or more of the following: an apta-DNA enzyme, an apta-ribozyme, an apta-MNAzyme.

28. The use according to claim 24, wherein: - the second target is an analyte; - the reaction mixture further comprises an oligonucleotide sequence capable of hybridising to a sensor arm of a second MNAzyme to thereby facilitate assembly of the second MNAzyme; - the second MNAzyme comprises an aptamer sequence capable of binding to the second target; and - Binding of the second target to the aptamer sequence of the second MNAzyme is capable of rendering the second MNAzyme catalytically active by facilitating the removal of an inhibitory molecule bound to the aptamer of the second MNAzyme.

29. Use according to claim 28, wherein the analyte is a molecule.

30. The use according to claim 28, wherein the analyte is a protein or a chemical compound.

31. The use of any one of claims 1 to 11, wherein the fluorophore of the first closed stem-loop oligonucleotide and the first open stem-loop oligonucleotide is the same as the fluorophore of the second closed stem-loop oligonucleotide and the second open stem-loop oligonucleotide.

32. The use according to claim 19, wherein: - the reaction mixture comprises a first MNAzyme and a second MNAzyme; and - the sequence of the loop portion of the first closed stem-loop oligonucleotide capable of hybridising to the substrate arm of the first MNAzyme is different from the sequence of the loop portion of the second closed stem-loop oligonucleotide capable of hybridising to the substrate arm of the second MNAzyme.

33. The use according to any one of claims 1 to 11, wherein the fluorophores of the first closed stem-loop oligonucleotide and the first open stem-loop oligonucleotide and the fluorophores of the second closed stem-loop oligonucleotide and the second open stem-loop oligonucleotide are detectable in a single fluorescence emission channel of a device.

34. The use according to any one of claims 1 to 11, wherein the enzyme does not induce cleavage or degradation of any of the targets or amplicons thereof.

35. The use according to any one of claims 1 to 11, wherein the first closed stem-loop oligonucleotide and the second closed stem-loop oligonucleotide each consist of the double-stranded stem portion and the single-stranded loop portion.

36. The use according to any one of claims 1 to 11, wherein the method further comprises determining the presence or absence of a third target or an amplicon thereof in the sample by: (d) contacting the reaction mixture comprising the sample with: a third closed stem-loop oligonucleotide, wherein the third closed stem-loop oligonucleotide comprises a double-stranded stem portion of hybridized nucleotides linked to a closed single-stranded loop portion of unhybridized nucleotides, wherein: the number of hybridizing nucleotides and / or the sequence of hybridizing nucleotides in the stem portion of the third closed stem-loop oligonucleotide is different from the number of hybridizing nucleotides and / or the sequence of hybridizing nucleotides in the stem portion of the first closed stem-loop oligonucleotide and the second closed stem-loop oligonucleotide; said closed single-stranded loop portion of said third closed stem-loop oligonucleotide is capable of hybridizing to a complementary sequence when in the presence of said target or an amplicon thereof; The double-stranded stem portion includes a fluorophore molecule attached to one strand and a quencher molecule attached to the opposite strand; and an enzyme capable of cleaving or degrading the single-stranded loop portion of the third closed stem-loop oligonucleotide only when in contact with the target or an amplicon thereof; (e) treating the reaction mixture: - under conditions suitable for said enzyme to induce cleavage or degradation of said loop portion of said third closed stem-loop oligonucleotide to thereby produce a third open stem-loop oligonucleotide; - at a third temperature, at or above the third temperature, the strands of the double-stranded stem portion of the third open stem-loop oligonucleotide dissociate to thereby promote spatial separation of the fluorophore molecule and the quencher molecule of the stem portion of the third open stem-loop oligonucleotide and provide a third detectable fluorescent signal; in: The third temperature is higher than the first temperature and the second temperature, and (f) detecting the levels of the first detectable fluorescent signal, the second detectable fluorescent signal, and the third detectable fluorescent signal at one or more temperatures, including or consisting of a temperature equal to or greater than the third temperature, to thereby determine the presence or absence of the target in the sample.

37. The use according to any one of claims 1 to 11, wherein the method further comprises determining the presence or absence of a third target or an amplicon thereof in the sample by: (d) contacting the reaction mixture comprising the sample with: a third closed stem-loop oligonucleotide, wherein the third closed stem-loop oligonucleotide comprises a double-stranded stem portion of hybridized nucleotides linked to a closed single-stranded loop portion of unhybridized nucleotides, wherein: the number of hybridizing nucleotides and / or the sequence of hybridizing nucleotides in the stem portion of the third closed stem-loop oligonucleotide is the same as the number of hybridizing nucleotides and / or the sequence of hybridizing nucleotides in the stem portion of the first closed stem-loop oligonucleotide or the second closed stem-loop oligonucleotide; said closed single-stranded loop portion of said third closed stem-loop oligonucleotide is capable of hybridizing to a complementary sequence when in the presence of said target or an amplicon thereof; the double-stranded stem portion comprising a fluorophore molecule attached to one strand and a quencher molecule attached to the opposite strand, wherein the fluorophore molecule attached to the third closed stem-loop oligonucleotide emits in a different color region of the visible spectrum than the fluorophore of the first closed stem-loop oligonucleotide and / or the second closed stem-loop oligonucleotide; and an enzyme capable of cleaving or degrading the single-stranded loop portion of the third closed stem-loop oligonucleotide only when in contact with the target or an amplicon thereof; (e) treating the reaction mixture: - under conditions suitable for said enzyme to induce cleavage or degradation of said loop portion of said third closed stem-loop oligonucleotide to thereby produce a third open stem-loop oligonucleotide; - at a third temperature, at which the strands of the double-stranded stem portion of the third open stem-loop oligonucleotide dissociate to thereby promote spatial separation of the fluorophore molecule and the quencher molecule of the stem portion of the third open stem-loop oligonucleotide and provide a third detectable fluorescent signal; (f) detecting the levels of the first detectable fluorescent signal, the second detectable fluorescent signal, and the third detectable fluorescent signal at one or more temperatures, including or consisting of a temperature equal to or greater than the third temperature, to thereby determine the presence or absence of the target in the sample.

38. Use according to claim 37, wherein the third temperature is different from the first temperature and / or the second temperature.

39. The use according to claim 38, wherein the method comprises treating the reaction mixture under conditions suitable for any one or more of the following: - a third MNAzyme binds to said third target or amplicon thereof and the substrate arm of said third MNAzyme hybridises to said loop portion of said third closed stem-loop oligonucleotide to thereby facilitate said cleavage of said loop portion of said third closed stem-loop oligonucleotide by said third MNAzyme to form said third open stem-loop oligonucleotide; - said third target or an amplicon thereof hybridizes to said loop portion of said third closed stem-loop oligonucleotide to form a double-stranded sequence for a third restriction endonuclease to associate with said double-stranded sequence and thereby promote said cleavage of said loop portion of said third closed stem-loop oligonucleotide, thereby forming said third open stem-loop oligonucleotide; - a third target or an amplicon thereof hybridizes to said loop portion of said third closed stem-loop oligonucleotide to form a third double-stranded sequence comprising said third target or an amplicon thereof, a third primer oligonucleotide hybridizing to the third target or an amplicon thereof to form a third double-stranded sequence, the third double-stranded sequence being located upstream (5') relative to the third double-stranded sequence including the third target or an amplicon thereof; a third enzyme having exonuclease activity associating with the loop portion of the third closed stem-loop oligonucleotide at or near an end of the third primer oligonucleotide, and catalytic activity of the third enzyme having exonuclease activity to thereby promote degradation of the loop portion of the third double-stranded sequence comprising the third target or amplicon and form the third open stem-loop oligonucleotide; - a third target or an amplicon thereof hybridizes to said loop portion of said third closed stem-loop oligonucleotide to form a third double-stranded sequence comprising said third target or an amplicon thereof, a third enzyme having exonuclease activity that associates with the third double-stranded sequence comprising the third target or an amplicon thereof, and catalytic activity of the third enzyme having exonuclease activity to thereby promote degradation of the loop portion of the third double-stranded sequence comprising the third target or amplicon and form the third open stem-loop oligonucleotide; in: The third target is a nucleic acid.

40. The use according to claim 36, wherein the treatment further comprises treating the reaction mixture under conditions suitable for: - the third cofactor binds to the DNA enzyme and / or the third cofactor binds to the ribozyme so that the DNA enzyme and / or the ribozyme exhibit catalytic activity, The DNA enzyme and / or ribozyme hybridizes to the loop portion of the third closed stem-loop oligonucleotide, the catalytic activity of the DNA enzyme and / or the ribozyme, thereby promoting the cleavage of the loop portion of the third closed stem-loop oligonucleotide and forming the third open stem-loop oligonucleotide, in: The third target is the third cofactor.

41. The use according to claim 40, wherein the cofactor is a metal ion.

42. The use according to claim 39, wherein the restriction endonuclease is a nicking endonuclease capable of associating with and cleaving the circular strand of the double-stranded sequence comprising the third target or an amplicon thereof.

43. The use of claim 39, wherein the nucleic acid sequence is capable of hybridising to a sensor arm of the third MNAzyme to thereby facilitate assembly of the third MNAzyme.

44. The use according to claim 36, wherein: - said third target is an analyte; - the enzyme comprises an enzyme having an aptamer, the aptamer being capable of binding to the third target; and - The binding of the third target to the aptamer can cause the enzyme having the aptamer to exhibit catalytic activity.

45. Use according to claim 44, wherein the analyte is a molecule.

46. ​​The use according to claim 44, wherein the analyte is a protein or a compound.

47. The use according to claim 44, wherein the enzyme having an aptamer comprises any one or more of the following: an apta-DNA enzyme, an apta-ribozyme, an apta-MNAzyme.

48. The use according to claim 39, wherein: - said third target is an analyte; - the reaction mixture further comprises an oligonucleotide sequence capable of hybridising to the sensor arm of the third MNAzyme to thereby facilitate assembly of the third MNAzyme; - said third MNAzyme comprises an aptamer sequence capable of binding to said third target; and - Binding of the third target to the aptamer sequence of the third MNAzyme is capable of rendering the third MNAzyme catalytically active by facilitating the removal of an inhibitory molecule bound to the aptamer of the third MNAzyme.

49. Use according to claim 48, wherein the analyte is a molecule.

50. The use according to claim 48, wherein the analyte is a protein or a chemical compound.

51. The use according to claim 39, wherein: - the reaction mixture comprises the third MNAzyme and either or both of the first MNAzyme defined in claim 3 and the second MNAzyme defined in claim 19; and - the sequence of the loop portion of the third closed stem-loop oligonucleotide capable of hybridising to the substrate arm of the third MNAzyme is different from: the sequence of the loop portion of the first closed stem-loop oligonucleotide capable of hybridizing to the substrate arm of the first MNAzyme, and / or The sequence of the loop portion of the second closed stem-loop oligonucleotide is capable of hybridising to the substrate arm of the first MNAzyme.

52. The use according to claim 36, wherein: - the number of hybridizing nucleotides and / or the sequence of hybridizing nucleotides in the stem portion of the third closed stem-loop oligonucleotide is different from the number of hybridizing nucleotides and / or the sequence of hybridizing nucleotides in the stem portion of the first closed stem-loop oligonucleotide and the second closed stem-loop oligonucleotide; - the third temperature is different from the first temperature and the second temperature; and - the fluorophores of the third closed stem-loop oligonucleotide and the third open stem-loop oligonucleotide emit in the same color region of the visible spectrum as the fluorophores of the first closed stem-loop oligonucleotide and the second closed stem-loop oligonucleotide and the first open stem-loop oligonucleotide and the second open stem-loop oligonucleotide.

53. The use according to claim 36, wherein: The fluorophore of the third closed stem-loop oligonucleotide and the third open stem-loop oligonucleotide is the same as the fluorophore of the first closed stem-loop oligonucleotide and / or the second closed stem-loop oligonucleotide and the first open stem-loop oligonucleotide and / or the second open stem-loop oligonucleotide.

54. The use according to claim 53, wherein the fluorophores of the third closed stem-loop oligonucleotide and the third open stem-loop oligonucleotide and the fluorophores of the first closed stem-loop oligonucleotide and the first open stem-loop oligonucleotide and / or the fluorophores of the second closed stem-loop oligonucleotide and the second open stem-loop oligonucleotide can be detected using the same fluorescence emission channel of the device.

55. The use according to claim 36, wherein: the fluorophores of the first closed stem-loop oligonucleotide and the first open stem-loop oligonucleotide emit in the same color region of the visible spectrum as the fluorophores of the second closed stem-loop oligonucleotide and the second open stem-loop oligonucleotide; and - the fluorophores of the third closed stem-loop oligonucleotide and the third open stem-loop oligonucleotide emit in a different color region of the visible spectrum than the fluorophores of the first closed stem-loop oligonucleotide and the second closed stem-loop oligonucleotide and the first open stem-loop oligonucleotide and the second open stem-loop oligonucleotide.

56. The use of claim 36, wherein the third temperature differs from the first temperature and / or the second temperature by more than: 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C.

57. The use according to claim 36, wherein detection of a third fluorescent signal at the third temperature indicates the presence of the third target in the sample, and failure to detect a third fluorescent signal at the third temperature indicates the absence of the third target in the sample.

58. The use according to any one of claims 1 to 11, wherein: (i) determining that the presence of the first detectable fluorescent signal at the first temperature indicates the presence of the first target in the sample, and determining that the absence of the first detectable fluorescent signal indicates the absence of the first target in the sample; and (ii) determining that the presence of the second detectable fluorescent signal at the second temperature indicates the presence of the second target in the sample, and determining that the absence of the second detectable fluorescent signal indicates the absence of the second target in the sample.

59. The use of any one of claims 1 to 11, wherein the determining the presence or absence of the first target and the second target comprises melting curve analysis using the first detectable fluorescent signal and the second detectable fluorescent signal.

60. The use of any one of claims 1 to 11, wherein part (c) comprises detecting the levels of the first detectable fluorescent signal and the second detectable fluorescent signal at a temperature of: - a temperature equal to or higher than said second temperature; and - a temperature that is equal to or higher than the first temperature and lower than the second temperature.

61. The use of claim 60, wherein part (c) further comprises detecting the levels of the first detectable fluorescent signal and the second detectable fluorescent signal at a temperature lower than the second temperature.

62. The use of claim 60, wherein part (c) comprises detecting the level of the first detectable fluorescent signal and the second detectable fluorescent signal during and / or after completion of the nucleic acid amplification reaction.

63. The use according to claim 60, wherein the method further comprises generating a first target positive control fluorescent signal using a known concentration of the first target and / or a known concentration of the first closed stem-loop oligonucleotide.

64. The use of claim 60, wherein the method further comprises generating a first target positive control fluorescent signal by repeating the method on a separate control sample comprising the first target.

65. The use according to claim 64, wherein the control sample comprising the first target comprises a known concentration of the first target.

66. The use of claim 64, wherein the control sample comprising the first target further comprises the second target.

67. The use of claim 59, wherein the method further comprises generating a second target positive control fluorescent signal by repeating the method on a separate control sample comprising the second target.

68. The use according to claim 67, wherein the control sample comprising the second target comprises a known concentration of the second target.

69. The use of claim 67 or claim 68, wherein the control sample further comprises the first target.

70. The use of claim 57, wherein the method further comprises generating a combined positive control fluorescent signal by repeating the method on a separate control sample comprising the first target and the second target.

71. The use of claim 70, wherein the combined control sample comprises a known concentration of the first target and / or a known concentration of the second target.

72. The use of claim 63, wherein the method further comprises normalizing the first detectable fluorescent signal and / or the second detectable fluorescent signal using any of the positive control fluorescent signals.

73. The use according to claim 59, wherein the method further comprises generating a negative control fluorescent signal by repeating the method as described in claim 1 on a separate negative control sample that does not contain: (i) the first target; or (ii) the second target; or (iii) the first target or the second target.

74. The use of claim 73, wherein the method further comprises normalizing the first detectable fluorescent signal and / or the second detectable fluorescent signal using the negative control fluorescent signal.

75. The use of claim 59, wherein the method further comprises comparing the first detectable fluorescent signal and / or the second detectable fluorescent signal to a threshold value, wherein: The threshold is generated using fluorescence signals obtained from a series of samples tested according to the method described in claim 1 and including any one or more of the following: (i) No template control and the first target (ii) No template control and the second target (iii) No template control, the first target and the second target To thereby determine the presence or absence of the first target and the second target in the sample.

76. The use according to claim 75, wherein the series of samples are tested using a known concentration of the first closed stem-loop oligonucleotide and / or a known concentration of the second closed stem-loop oligonucleotide.

77. The use of any one of claims 1 to 11, wherein the first temperature differs from the second temperature by more than: 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C or 80°C.

78. The use according to any one of claims 1 to 11, wherein any of the amplicons thereof are produced by any one or more of the following: polymerase chain reaction (PCR), strand displacement amplification (SDA), helicase-dependent amplification (HDA), recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), rolling circle amplification (RCA), transcription-mediated amplification (TMA), self-sustained sequence replication (3SR), nucleic acid sequence-based amplification (NASBA) and / or reverse transcription polymerase chain reaction (RT-PCR).

79. The use according to any one of claims 1 to 11, wherein the sample is a biological sample obtained from a subject.

80. The use according to any one of claims 1 to 11, wherein the method is performed in vitro.

81. The use according to any one of claims 1 to 11, wherein the method is performed ex vivo.

82. A composition comprising: (i) a first closed stem-loop oligonucleotide and a second closed stem-loop oligonucleotide, wherein each of the closed stem-loop oligonucleotides comprises a double-stranded stem portion of hybridized nucleotides linked to a single-stranded loop portion of unhybridized nucleotides, wherein: The first closed stem-loop oligonucleotide and the second closed stem-loop oligonucleotide differ in the number of hybridizing nucleotides and / or the sequence of hybridizing nucleotides in the stem portion, Each of the double-stranded stem portions comprises a fluorophore molecule attached to one strand and a quencher molecule attached to the opposite strand, and The single-stranded loop portion includes a region capable of serving as a substrate for a catalytic nucleic acid, exonuclease, or endonuclease; wherein the fluorophore molecules emit in the same color region of the visible spectrum, and the melting temperature (Tm) of the double-stranded stem portion of the first closed stem-loop oligonucleotide is different from the Tm of the double-stranded stem portion of the second closed stem-loop oligonucleotide; and (ii) an enzyme, wherein the enzyme is selected from a catalytic nucleic acid, an exonuclease or an endonuclease.

83. The composition of claim 82, wherein the enzyme is selected from an MNAzyme, a DNA enzyme, a ribozyme, an aptazyme, a restriction endonuclease, a nicking endonuclease, or a polymerase with exonuclease activity.

84. The composition of claim 82, wherein the single-stranded loop portion of the first closed stem-loop oligonucleotide is different in sequence from the single-stranded loop portion of the second closed stem-loop oligonucleotide.

85. The composition of claim 82, further comprising: a first MNAzyme comprising a substrate arm capable of hybridizing to the closed single stranded loop portion of the first closed stem-loop oligonucleotide; as well as A second MNAzyme comprising a substrate arm capable of hybridising to the single stranded loop portion of the second closed stem-loop oligonucleotide.

86. The composition of claim 85, wherein: the substrate arm of the first MNAzyme hybridizes to the single stranded loop portion of the first closed stem-loop oligonucleotide; and The substrate arm of the second MNAzyme hybridizes to the single stranded loop portion of the second closed stem-loop oligonucleotide.

87. The composition of claim 86, wherein: the first MNAzyme and / or the second MNAzyme comprises an aptamer sequence that binds to a target analyte and a sensor arm that hybridizes to an oligonucleotide sequence; and The first MNAzyme is designed to detect a different target than the second MNAzyme.

88. The composition of claim 87, wherein the analyte is a molecule.

89. The composition of claim 87, wherein the analyte is a protein or a compound.

90. The composition of claim 86, wherein: The first MNAzyme and / or the second MNAzyme comprises a sensor arm that hybridizes to a target sequence, and the first MNAzyme is designed to detect a different target than the second MNAzyme.

91. The composition of any one of claims 82 to 90, further comprising: a first oligonucleotide sequence that hybridizes to the single-stranded loop portion of the first closed stem-loop oligonucleotide that is different in sequence from the single-stranded loop portion of the second closed stem-loop oligonucleotide, thereby forming a double-stranded sequence including the first oligonucleotide; a second oligonucleotide sequence that hybridizes to the single-stranded loop portion of the second closed stem-loop oligonucleotide that is different in sequence from the single-stranded loop portion of the first closed stem-loop oligonucleotide, thereby forming a double-stranded sequence including the second oligonucleotide; a restriction endonuclease that associates with and is capable of cleaving the double-stranded sequence comprising the first oligonucleotide; and / or A restriction endonuclease that associates with and is capable of cleaving the double-stranded sequence comprising the second oligonucleotide.

92. The composition of any one of claims 82 to 90, further comprising: a first double-stranded sequence comprising a first target oligonucleotide sequence that hybridizes to the single-stranded loop portion of the first closed stem-loop oligonucleotide having a sequence different from that of the single-stranded loop portion of the second closed stem-loop oligonucleotide, thereby forming a first double-stranded sequence comprising the first target oligonucleotide; a second double-stranded sequence comprising a second target oligonucleotide sequence that hybridizes to the single-stranded loop portion of the second closed stem-loop oligonucleotide having a sequence different from that of the single-stranded loop portion of the first closed stem-loop oligonucleotide, thereby forming a double-stranded sequence comprising the second target oligonucleotide; a first primer oligonucleotide that hybridizes to the first target oligonucleotide upstream (5') relative to the first double-stranded sequence, and a first enzyme having exonuclease activity that associates with the loop portion of the first closed stem-loop oligonucleotide at or near an end of the first primer oligonucleotide; and / or a second primer oligonucleotide that hybridizes to the second target oligonucleotide upstream (5') relative to the second double-stranded sequence, and a second enzyme having exonuclease activity that associates with the loop portion of the second closed stem-loop oligonucleotide at or near an end of the second primer oligonucleotide.

93. The composition of any one of claims 82 to 90, further comprising: a first double-stranded sequence comprising a first target oligonucleotide sequence that hybridizes to the single-stranded loop portion of the closed first stem-loop oligonucleotide having a sequence different from that of the single-stranded loop portion of the second closed stem-loop oligonucleotide, thereby forming a first double-stranded sequence comprising the first target oligonucleotide; a second double-stranded sequence comprising a second target oligonucleotide sequence that hybridizes to the single-stranded loop portion of the second closed stem-loop oligonucleotide having a sequence different from that of the single-stranded loop portion of the first closed stem-loop oligonucleotide, thereby forming a second double-stranded sequence comprising the second target oligonucleotide; A first enzyme having exonuclease activity associates with the first double-stranded sequence, and a second enzyme having exonuclease activity associates with the second double-stranded sequence.

94. The composition of any one of claims 82 to 90, further comprising: a first DNA enzyme and / or a first ribozyme that hybridizes to a loop portion of the first closed stem-loop oligonucleotide that has a sequence different from that of the single-stranded loop portion of the second closed stem-loop oligonucleotide, a second DNA enzyme and / or a second ribozyme that hybridizes to the loop portion of the second closed stem-loop oligonucleotide having a sequence different from that of the single-stranded loop portion of the first closed stem-loop oligonucleotide, a cofactor for each of said DNA enzymes, said cofactor being capable of rendering each of said DNA enzymes catalytically active and thereby inducing the cleavage activity of any of said DNA enzymes hybridized to said loop portion, a cofactor for each of said ribozymes, said cofactor being capable of rendering each of said ribozymes catalytically active and thereby inducing the cleavage activity of any of said ribozymes hybridized to said loop portion.

95. The composition of any one of claims 82 to 90, further comprising: a third closed stem-loop oligonucleotide, wherein the third closed stem-loop oligonucleotide comprises a double-stranded stem portion of hybridized nucleotides linked to a single-stranded loop portion of unhybridized nucleotides, wherein: The number of hybridizing nucleotides and / or the sequence of hybridizing nucleotides in the stem portion of the third closed stem-loop oligonucleotide is different from the number of hybridizing nucleotides and / or the sequence of hybridizing nucleotides in the stem portion of the first closed stem-loop oligonucleotide and the second closed stem-loop oligonucleotide, and The double-stranded stem portion of the third closed stem-loop oligonucleotide comprises a fluorophore molecule attached to one strand and a quencher molecule attached to the opposite strand, wherein the fluorophore molecule of the third closed stem-loop oligonucleotide emits in a different color region of the visible spectrum than the fluorophore molecules of the first closed stem-loop oligonucleotide and the second closed stem-loop oligonucleotide.

96. The composition of any one of claims 82 to 90, further comprising: a third closed stem-loop oligonucleotide, wherein the third closed stem-loop oligonucleotide comprises a double-stranded stem portion of hybridized nucleotides linked to a single-stranded loop portion of unhybridized nucleotides, wherein: The number of hybridizing nucleotides and / or the sequence of hybridizing nucleotides in the stem portion of the third closed stem-loop oligonucleotide is different from the number of hybridizing nucleotides and / or the sequence of hybridizing nucleotides in the stem portion of the first closed stem-loop oligonucleotide and the second closed stem-loop oligonucleotide, and The double-stranded stem portion of the third closed stem-loop oligonucleotide comprises a fluorophore molecule attached to one strand and a quencher molecule attached to the opposite strand, wherein the fluorophore molecule of the third closed stem-loop oligonucleotide emits in the same color region of the visible spectrum as the fluorophore molecules of the first closed stem-loop oligonucleotide and the second closed stem-loop oligonucleotide.

97. The composition of any one of claims 82 to 90, further comprising: a third closed stem-loop oligonucleotide, wherein the third closed stem-loop oligonucleotide comprises a double-stranded stem portion of hybridized nucleotides linked to a single-stranded loop portion of unhybridized nucleotides, wherein: The number of hybridizing nucleotides and / or the sequence of hybridizing nucleotides in the stem portion of the third closed stem-loop oligonucleotide is the same as the number of hybridizing nucleotides and / or the sequence of hybridizing nucleotides in the stem portion of the first closed stem-loop oligonucleotide or the second closed stem-loop oligonucleotide, and The double-stranded stem portion of the third closed stem-loop oligonucleotide comprises a fluorophore molecule attached to one strand and a quencher molecule attached to the opposite strand, wherein the fluorophore molecule of the third closed stem-loop oligonucleotide emits in a different color region of the visible spectrum than the fluorophore molecules of the first closed stem-loop oligonucleotide and the second closed stem-loop oligonucleotide.

Images