RU2783946C2 - Multiplex detection of nucleic acids - Google Patents

Abstract

FIELD: molecular biology.

SUBSTANCE: group of inventions specifically relates to oligonucleotides and a method for their use for detection and/or differentiation of target nucleic acids. A method for determination of the presence or absence of the first and the second targets in a sample includes obtaining a reaction mixture by bringing the sample or its derivative presumably containing the first and/or the second targets or their amplicons into contact with the first and the second closed oligonucleotides of a “stem-loop” type; treatment of the reaction mixture, detection of levels of the first and the second fluorescent signals at one or more temperatures, which allows for determination of the presence or absence of the specified targets in the sample. A composition for determination of the presence or absence of the first and the second targets in the sample contains the first and the second closed oligonucleotides of a “stem-loop” type.

EFFECT: group of inventions provides for expansion of possibilities of instruments for differentiation of several targets, using one type of a detector, for example, one fluorescence channel.

81 cl, 28 dwg, 19 tbl, 14 ex

Description

Technical field

The present invention generally relates to the field of molecular biology. More specifically, the present invention provides oligonucleotides and methods for using them to detect and/or differentiate target nucleic acids. These oligonucleotides and methods are of particular use in the amplification, detection and/or discrimination of multiple targets simultaneously.

Inclusion via cross-reference

This application claims the priority of Australian Provisional Application No. 2018902915; filed August 9, 2018, the contents of which are incorporated herein by cross-reference in their entirety.

State of the art

Genetic analysis is becoming a routine procedure in the clinical setting to assess disease risk, diagnose disease, establish patient prognosis or patient response to therapy, and monitor patient progress. The implementation of such genetic assays depends on the development of simple, inexpensive and rapid assay methods for recognizing genetic changes.

Nucleic acid amplification methods in vitro are widely used in genetics and disease diagnostics. Such methods include polymerase chain reaction (PCR), strand displacement amplification (SDA), helicase dependent amplification (HDA), recombinase polymerase amplification (RPA), loop isothermal amplification (LAMP), rolling ring 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 exponential amplification of amplicons containing oligonucleotide primers at the 5' ends and newly synthesized copies of the sequences between the primers.

Commonly used methods for monitoring amplicon accumulation in real time or after amplification is complete include detection using MNCzymes with universal substrate probes; use of target-specific molecular beacons, TaqMan probes or hydrolysis probes; primers/probes of the "scorpion" type; and/or the use of intercalating dyes such as SybGreen. High resolution melt curve analysis can be performed during or after several of these protocols to provide additional information, as amplicons with different sequences denature at different temperatures, known as melting point or Tm. Such protocols measure the melting curves that result from a) separation of two strands of double-stranded amplicons in the presence of an intercalating dye, or b) separation of one strand of an amplicon and a complementary target-specific probe labeled with a fluorophore and quencher. Analysis of the melting curve provides information on the kinetics of dissociation of two DNA strands during heating. The melting point (Tm) is the temperature at which 50% of the DNA dissociates. Tm depends on the length, composition of the sequence, and the content of G-C in the paired nucleotides. Elucidation of target DNA information from melt curve analysis typically involves a series of fluorescence measurements at short intervals, usually over a wide range of temperatures. The melting point depends not only on the sequence of bases. The melting temperature can be affected by the concentrations of oligonucleotides, cations in the buffer (both monovalent (Na+) and divalent (Mg2+) salts), and the presence or absence of destabilizing agents, such as urea or formamide.

In general, the number of available fluorescent channels capable of controlling individual wavelengths limits the number of targets that can be detected and specifically detected in a single reaction on a real-time instrument. A recently developed protocol known as Tagging Oligonucleotide Cleavage and Extension (TOCE) extends this capability by allowing multiple targets to be analyzed at a single wavelength. The TOCE technology uses "feeding" and "receiving" oligonucleotides (Pitcher and Catcher). "Feed" oligonucleotides contain two regions: a targeting fragment complementary to the target, and a labeled fragment that is not complementary and located at the 5' end.

The capture oligonucleotide contains a double tag and a region at the 3' end that is complementary to the labeled fragment of the "feed" oligonucleotide. During amplification, the "feed" oligonucleotide binds to the amplicons, and when the primer is extended, the exonuclease activity of the polymerase can cleave the labeled fragment from the "feed" oligonucleotide. The released labeled fragment then binds to the "receiving" oligonucleotide and acts as a primer for the synthesis of the complementary strand. Therefore, the melting point of the double-stranded host oligonucleotide molecule (Catcher-Tm) is used as a surrogate marker for the parent template. Because multiple "receiver" oligonucleotides can be used, having different sequences and lengths and melting at different temperatures, it is possible to obtain a series of Catcher-Tm values indicating a series of targets despite being measured at the same wavelength. Limitations of this approach include the inherent complexity of requiring the released fragment to initiate and complete a second chain extension on an artificial target.

Hairpin or stem-loop probes have also proven to be useful tools for nucleic acid detection and/or target amplification monitoring. Hairpin probes doubly labeled with a pair of fluorophore/quencher dyes are commonly known in the art as molecular beacons. In general, these molecules have three features; 1) a stem structure formed by hybridization of the complementary 5'- and 3'-ends of the oligonucleotide; 2) a loop region complementary to the target or target amplicon to be detected; and 3) a pair of fluorophore/quencher dyes attached to the ends of the molecular beacon. During PCR, the loop region binds to amplicons by complementarity and this results in stem melting and separation of the fluorophore/quencher dye pair. Separation of a pair of dyes attached to the ends of an intact open molecular beacon causes a change in fluorescence that indicates the presence of a target. This method is commonly used for multiplexing multiple targets in a single PCR assay. In multiplex reactions, each molecular beacon contains its own target-specific loop region and a unique fluorophore, so that hybridization of each molecular beacon with each amplicon species can be monitored on a separate channel, i.e. at a particular wavelength.

The concept of molecular beacons is expanded in a strategy known as "low specificity beacons" (Sloppy Beacons). In this protocol, the loop region of a single beacon is long enough to tolerate base mismatch and therefore binds to a number of closely related targets that differ in one or more nucleotides. After amplification, a melting curve analysis is performed, which makes it possible to differentiate different types of targets depending on the temperature at which the target and the beacon loop separate (melt). Thus, it is possible to simultaneously detect and distinguish between several closely related species at the same wavelength by determining the melting profile of specific targets with a single low specific beacon. Standard molecular beacons and low specific beacons differ from TaqMan probes and hydrolysis probes in that they are not designed to be destroyed or cleaved during amplification. A disadvantage of DNA hybridization-based technologies, such as low-specificity beacons and TOCE, is the potential for false positive results due to non-specific hybridization between probes and non-specific nucleotide sequences.

Many nucleic acid detection assays use melt curve analysis to detect the presence of specific target sequences in a given sample. Melting curve analysis protocols include the measurement of fluorescence at various temperatures over a gradually increasing temperature range. The slope of this curve is then plotted as a function of temperature to obtain a melting curve. This process is often slow and usually takes 30 to 60 minutes. In addition, melt curve analysis may require interpretation by a qualified person and/or the use of special software to interpret the results. Consequently, there is a strong demand for faster and/or simpler alternatives to melt curve analysis.

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 alternative target amplification protocols.

The essence of the invention

The present invention addresses one or more of the shortcomings of current assays involving multiplex detection.

The present invention provides methods and compositions that enhance multiplexing during amplification protocols using oligonucleotide structures, herein referred to as LOCS ( Lo ops Connected to S tems - loops connected to stems). A series of LOCS labeled with the same fluorophore/quencher pair can be individually distinguished within a single reaction based on the temperature at which the stem fragment melts after the LOCS is opened by cleavage or disruption in response to the presence of a target. Thus, the melting point of the stem region is used as a surrogate marker for the specific target that caused the LOCS to open. While other methods involving the use of a stem-loop structure use a change in fluorescence after a) hybridization of the loop region with target amplicons (molecular beacons and low-specificity beacons), increasing the distance between dye pairs, or b) cleavage, providing physical separation of dyes (cleavable molecular beacons), the present invention offers improvements over existing assays for multiplex detection. These improvements are due, at least in part, to controlling the melting temperature of the stem fragment of the stem-loop oligonucleotides by changing the length and/or composition of the stem sequence so that each stem melts at its own temperature.

As described herein, multiple LOCS labeled with the same fluorophore may contain a) different loop sequences, allowing direct or indirect detection of multiple targets simultaneously, and b) different stem sequences melting at different temperatures that can be used. to identify a specific target(s) present(s) in several test objects. The methods of the present invention provide one or more advantages over methods known in the art, such as the TOCE protocol, in that there is no need for separate "acceptor" molecules, which reduces the number of components in the reaction mixture and reduces costs. Moreover, the TOCE method is inherently more complex than the methods of the present invention because it requires the released fragment to initiate and complete the second extension on the synthetic target. In addition, in some embodiments, LOCS probes can be versatile (regardless of the target sequence) and/or can be combined with a variety of detection technologies, providing broad applicability in the field of molecular diagnostics. In addition, the melting temperature used in conventional amplification and detection methods is based on hybridization and melting of the probe with the target nucleic acid. The disadvantage of these methods is an increase in the number of false positives due to non-specific hybridization between probes and non-specific nucleotide sequences. The methods of the present invention overcome this limitation because LOCS reporter probes containing universal substrates do not bind to the target sequence. Finally, it is well known in the art that intramolecular bonds are stronger than intermolecular bonds, and hence the likelihood of these uncleaved (closed) LOCS hybridizing to a non-specific target and producing a false positive signal is much lower.

As a result of intramolecular bonds being stronger than intermolecular bonds, doubly labeled LOCS melts at the same temperature in the intact (closed) state and at a lower temperature after opening the loop region through target-dependent cleavage or degradation. This property of nucleic acids is used in the present invention to enhance the ability of tools to distinguish between multiple targets using a single type of detector, such as a single fluorescence channel.

The temperature dependent fluorescence signals produced by the LOCS reporters of the present invention are well defined and independent of the target DNA. Thus, it is possible to obtain information about the target DNA based on measurements of the fluorescence signal generated at selected temperatures rather than over the entire temperature gradient, which has the advantage of reducing the operating time of thermal cyclers (eg, PCR devices). As a non-limiting example, on a Bio-Rad CFX96 PCR system, performing a traditional melt assay with temperature settings from 20°C to 90°C in 0.5°C increments and a dwell time of 5 seconds requires 141 fluorescence measurement cycles, which takes approximately 50 minutes. When using LOCS probes, target DNA information can be obtained on the same device in 2-6 fluorescence measurements, which takes approximately 2-5 minutes. Without particular limitation, reduced runtime can be beneficial in many applications, including, for example, diagnostics.

The LOCS probes of the present invention can also be used to simultaneously detect, differentiate and/or quantify multiple targets on a single fluorescence channel. In conventional qPCR, the target DNA is quantitated using the quantification cycle value (Cq) based on the amplification curve obtained by measuring fluorescence at the same temperature in each amplification cycle. The value of Cq is proportional to the negative logarithmic value of the concentration of the target DNA, and therefore this concentration can be determined from the experimentally determined value of Cq. However, if there is more than one target-specific probe in the same channel, the correct and specific quantification of each target is difficult, since it is difficult to determine whether the signal comes from a particular probe. By solving this problem, LOCS allows more than one target to be correctly and accurately quantified in one channel, provided that the amplification curve is obtained by measuring fluorescence during amplification at more than one temperature. This is possible because different LOCS can produce significantly different fluorescence at different temperatures.

In some embodiments where the assay requires fluorescence acquisition only at a limited number of time points during PCR, such as after PCR, the use of LOCS structures eliminates the need to acquire data in each run. Thus, these implementations are well-suited for protocols with very fast cycle times, which can reduce result time.

As noted above, melting curve analysis protocols involve measuring fluorescence at various temperatures over a gradually increasing temperature range (eg, between 30°C and 90°C). The slope of this curve is then plotted as a function of temperature to obtain a melting curve. This process is often slow and may take, for example, 30 to 60 minutes. Increasing the speed of melt curve analysis requires access to highly specialized instruments and is not achievable with standard PCR devices. Thus, there is a great need for faster alternatives to melt curve analysis that can provide simultaneous detection of multiple targets on a single fluorescence channel using standard instruments. The melting point (Tm) of the LOCS structures of the present invention is predetermined and constant (ie, independent of the target sequence or its concentration), and therefore does not require gradual change over the entire temperature gradient range. Each LOCS structure requires only one fluorescence measurement at its specific Tm, which eliminates the need for a full temperature gradient, speeds up the result, and therefore overcomes the above limitations.

In addition, melt curve analysis usually requires interpretation by qualified personnel or the use of specialized software to interpret the results.

In some embodiments of the present invention, the measurement of fluorescence at a single temperature after completion of PCR eliminates the need for subjective interpretation of melting curves and facilitates objective determination of the presence or absence of targets.

In other embodiments of the present invention, the assay may require fluorescence to be recorded only at a limited number of time points within a PCR, such as post-PCR, eliminating the need to record data in each run. Thus, these implementations are well-suited for protocols with very fast cycle times, which can reduce result time.

Several methods have been described that involve recording fluorescence at multiple temperatures during PCR, including recording at two temperatures, facilitating the distinction between perfectly matched and mismatched probes. In addition, some protocols use multiple detection temperatures after each PCR cycle to quantify the concentration of each target when two targets are present and detected on the same channel. Other methods for simultaneous quantification of two targets are achieved by plotting a complete melting curve at the end of each PCR cycle.

The LOCS structures of the present invention may be compatible with most (and potentially all) of these existing assays.

The present invention relates at least in part to the following embodiments 1-59:

Embodiment 1. A method for determining the presence or absence of first and second targets in a sample, said method comprising:

- obtaining a reaction mixture by bringing the sample or its derivative, presumably containing the first and/or second targets or their amplicons, into contact with:

the first and second closed stem-loop oligonucleotides, each of the closed stem-loop oligonucleotides containing a double-stranded stem fragment formed by hybridized nucleotides and connected to a closed single-stranded loop fragment formed by unhybridized nucleotides, wherein:

the number of hybridized nucleotides and/or the sequence of hybridized nucleotides in the stem fragment of the first and second closed stem-loop oligonucleotides are different, and

each of the double-stranded stem fragments contains a fluorophore molecule attached to one strand and a quencher molecule attached to the opposite strand; and

enzymes capable of cleaving or destroying single-stranded loop fragments of the first and second closed stem-loop oligonucleotides only upon contact with the target or its amplicon;

- processing of the reaction mixture:

under conditions suitable for the enzymes to induce cleavage or degradation of the loop fragment of the first and second closed stem-loop oligonucleotides, thereby forming the first and second open stem-loop oligonucleotides;

- detecting the presence or absence of said first and second disclosed oligonucleotides by treating the reaction mixture or a derivative thereof at: a first temperature at which the chains of the double-stranded stem fragment of the first disclosed stem-loop oligonucleotide dissociate, thereby facilitating the spatial separation of the fluorophore and stem quencher molecules fragment of the first disclosed stem-loop oligonucleotide and providing a first detectable signal, and

a second temperature at which the strands of the double-stranded stem fragment of the second disclosed stem-loop oligonucleotide dissociate, thereby facilitating the spatial separation of the fluorophore and quencher molecules of the stem fragment of the second disclosed stem-loop oligonucleotide and providing a second detectable signal,

and:

the first temperature is different from the second temperature,

the fluorophore of the first disclosed stem-loop oligonucleotide and the fluorophore of the second disclosed stem-loop oligonucleotide emit in the same color region of the visible spectrum, and

detection of the signal at the first temperature indicates the presence of the first target in the sample, and the inability to detect the signal at the first temperature indicates the absence of the first target in the sample; and

detection of a signal at the second temperature indicates the presence of the second target in the sample, and failure to detect the signal at the second temperature indicates the absence of the second target in the sample.

Embodiment 2. The method of Embodiment 1 wherein the enzymes contain multicomponent nucleozymes (MNCzymes) and said treatment of the reaction mixture comprises treating the reaction mixture under conditions suitable for:

binding a first multicomponent nucleo- zyme (MNKzyme) to a first target or amplicon thereof; and hybridizing the substrate strands of said first MNKzyme with a loop fragment of the first closed stem-loop oligonucleotide, thereby facilitating said cleavage of the loop fragment of the first closed stem-loop oligonucleotide by the first MNKzyme to form the first open stem-loop oligonucleotide.

Embodiment 3. The method of Embodiment 2 wherein the target is a nucleotide sequence or amplicon thereof capable of hybridizing to the sensor strands of the first MNKzyme, thereby facilitating assembly of the first MNKzyme.

Embodiment 4. The method of Embodiment 1, wherein:

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

- enzymes include enzymes with an aptamer capable of binding to the first target; and

binding of the first target to the aptamer is capable of making enzymes with the aptamer catalytically active.

Embodiment 5. The method of Embodiment 4 wherein the aptamer enzymes comprise any one or more of the following classes of compounds: apta-DNAzymes, apta-ribozymes, apta-MNAzymes.

Embodiment 6. A method according to any one of embodiments 2, 4, or 5, wherein:

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

- the reaction mixture additionally contains an oligonucleotide sequence capable of hybridizing with the sensor chains of the first MNKzyme, thereby facilitating the assembly of the first MNKzyme;

the first MNKzyme contains an aptamer sequence capable of binding to the first target; and

- binding of the target to the aptamer can make the first MNKzyme catalytically active.

Embodiment 7. The method of any one of Embodiments 1-6 wherein the enzymes comprise restriction endonucleases and said treatment of the reaction mixture comprises:

processing the reaction mixture under conditions suitable for hybridizing the first target or its amplicon with the loop fragment of the first closed stem-loop oligonucleotide to form a double-stranded sequence for binding the first restriction endonuclease, which facilitates said cleavage of the loop fragment of the first closed stem-loop oligonucleotide to form the first disclosed stem-loop oligonucleotide.

Embodiment 8. The method of Embodiment 7, wherein the restriction endonuclease is a nicking endonuclease capable of binding to and cleaving the loop strand of said double-stranded sequence for the first restriction endonuclease.

Embodiment 9. The method of any one of Embodiments 1 to 8, wherein the enzymes have exonuclease activity (e.g., polymerase enzymes, exonucleases) and said treatment of the reaction mixture comprises:

processing the reaction mixture under conditions suitable for:

- hybridization of the first target or its amplicon with the loop fragment of the first closed stem-loop oligonucleotide to form the first double-stranded sequence containing the first target or its amplicon,

- hybridization of the first primer-oligonucleotide with the first target or its amplicon with the formation of the second double-stranded sequence located upstream (in the direction of 5') relative to the first double-stranded sequence containing the first target or its amplicon,

association of the first enzyme having exonuclease activity with the loop fragment of the first closed stem-loop oligonucleotide at or near the end of the first primer-oligonucleotide, and

- catalytic activity of the first enzyme having exonuclease activity, which facilitates the destruction of the loop fragment of the first double-stranded sequence containing the first target or amplicon, and the formation of the first open stem-loop oligonucleotide.

Embodiment 10. The method of any one of Embodiments 1-9 wherein the enzymes have exonuclease activity and said treatment of the reaction mixture comprises:

processing the reaction mixture under conditions suitable for:

- hybridization of the first target or its amplicon with the loop fragment of the first closed stem-loop oligonucleotide to form the first double-stranded sequence containing the first target or its amplicon,

- Association of the first enzyme with exonuclease activity, with the first double-stranded sequence containing the first target or its amplicon; and

- catalytic activity of the first enzyme having exonuclease activity, which facilitates the destruction of the loop fragment of the first double-stranded sequence containing the first target or amplicon, and the formation of the first open stem-loop oligonucleotide.

Embodiment 11. The method of any one of Embodiments 1 to 10, wherein the enzymes comprise DNAzymes and/or ribozymes requiring a first cofactor to exhibit catalytic activity, and said treatment of the reaction mixture comprises treating the reaction mixture under conditions suitable for:

- linking said first cofactor to a DNAzyme and/or linking said first cofactor to a ribozyme conferring catalytic activity on the DNAzyme and/or ribozyme,

- hybridization of the DNAzyme and/or ribozyme with the loop fragment of the first closed stem-loop oligonucleotide,

- catalytic activity of the DNAzyme and/or ribozyme to facilitate cleavage of the loop fragment of the first closed stem-loop oligonucleotide and the formation of the first open stem-loop oligonucleotide.

and:

the first target is the first cofactor.

Embodiment 12. The method of Embodiment 11 wherein the first cofactor is a metal ion (eg, Mg ²⁺ , Mn ²⁺ , Ca ²⁺ , Pb ²⁺ ).

Embodiment 13. The method of any one of Embodiments 1-12, wherein said treatment further comprises treating the reaction mixture under conditions suitable for any one or more of the following processes:

- binding of the second MNKzyme with the second target or its amplicon and hybridization of the substrate chains of the specified second MNKzyme with the loop fragment of the second closed stem-loop oligonucleotide, which facilitates the specified cleavage of the loop fragment of the second closed stem-loop oligonucleotide by the second MNKzyme with the formation of the second a disclosed stem-loop oligonucleotide;

- hybridization of the second target or its amplicon with the loop fragment of the second closed stem-loop oligonucleotide to form a double-stranded sequence for binding the second restriction endonuclease to associate with the second double-stranded sequence, which facilitates said cleavage of the loop fragment of the second closed stem-loop oligonucleotide to form a second open stem-loop oligonucleotide.

- hybridization of the second target or its amplicon with the loop fragment of the second closed stem-loop oligonucleotide to form a second double-stranded sequence containing the second target or its amplicon,

hybridization of the second primer-oligonucleotide with the second target or its amplicon to form a second double-stranded sequence located upstream (in the 5' direction) relative to the second double-stranded sequence containing the second target or its amplicon,

association of the second enzyme having exonuclease activity with the loop fragment of the second closed stem-loop oligonucleotide at or near the end of the second primer-oligonucleotide, and

catalytic activity of a second enzyme having exonuclease activity, which facilitates the destruction of the loop fragment of the second double-stranded sequence containing the second target or amplicon, and the formation of the second open stem-loop oligonucleotide;

- hybridization of the second target or its amplicon with the loop fragment of the second closed stem-loop oligonucleotide to form a second double-stranded sequence containing the second target or its amplicon,

association of the second enzyme having exonuclease activity with the second double-stranded sequence containing the second target or its amplicon and the catalytic activity of the second enzyme having exonuclease activity, which facilitates the destruction of the loop fragment of the second double-stranded sequence containing the second target or amplicon and the formation of the second opened oligonucleotide stem-loop type;

- binding of the second cofactor to the DNAzyme and/or binding of the second cofactor to the ribozyme conferring catalytic activity on the DNAzyme and/or ribozyme,

hybridization of the DNAzyme and/or ribozyme with the loop fragment of the second closed stem-loop oligonucleotide,

catalytic activity of the DNAzyme and/or ribozyme to facilitate cleavage of the loop fragment of the second closed stem-loop oligonucleotide and the formation of the second open stem-loop oligonucleotide.

and:

the second target is the second cofactor.

Embodiment 14. The method of Embodiment 13 wherein the second cofactor is a metal ion (eg, Mg ²⁺ , Mn ²⁺ , Ca ²⁺ , Pb ²⁺ ).

Embodiment 15. The method of Embodiment 13 wherein the second restriction endonuclease is a nicking endonuclease capable of associating with and cleaving the loop strand of said double-stranded sequence containing the second target or its amplicon.

Embodiment 16. The method of Embodiment 15, wherein the first and second restriction endonucleases are different types of restriction endonucleases.

Embodiment 17. The method of Embodiment 15, wherein the first and second restriction endonucleases are the same type of restriction endonucleases.

Embodiment 18. The method of Embodiment 13 wherein the second target is a nucleotide sequence or amplicon thereof capable of hybridizing to the sensor strands of the second MNKzyme, thereby facilitating assembly of the second MNKzyme.

Embodiment 19. The method of Embodiment 13, wherein:

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

- enzymes include enzymes with an aptamer capable of binding to a second target; and

binding of the second target to the aptamer is capable of making enzymes with the aptamer catalytically active.

Embodiment 20. The method of Embodiment 19 wherein the aptamer enzymes comprise any one or more of the following classes of compounds: apta-DNAzymes, apta-ribozymes, apta-MNKzymes.

Embodiment 21. The method of Embodiment 13, 19, or 20, wherein:

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

- the reaction mixture additionally contains an oligonucleotide sequence capable of hybridizing with the sensor chains of the second MNKzyme, thereby facilitating the assembly of the second MNKzyme;

the second MNKzyme contains an aptamer sequence capable of binding to the second target; and

- binding of the second target to the aptamer sequence of the second MNKzyme is able to make the second MNKzyme catalytically active, facilitating the removal of the inhibitory molecule associated with the aptamer of the second MNKzyme.

Embodiment 22. The method of any one of Embodiments 1-21, wherein the fluorophore of the first closed and opened stem-loop oligonucleotides is the same as the fluorophore of the second closed and opened stem-loop oligonucleotides.

Embodiment 23. A method according to any of embodiments 13-22, wherein:

- the reaction mixture contains the specified first and second MNCzymes; and

- the sequence of the loop fragment of the first closed stem-loop oligonucleotide capable of hybridizing with the substrate chains of the first MNCzyme differs from the sequence of the loop fragment of the second closed stem-loop oligonucleotide capable of hybridizing with the substrate chains of the second MNCzyme.

Embodiment 24. The method of any one of embodiments 1-23, wherein the fluorophore of the first closed and opened stem-loop oligonucleotides and the fluorophore of the second closed and opened stem-loop oligonucleotides are detected on the same fluorescent emission channel of the device.

Embodiment 25. The method of any one of Embodiments 1-24, further comprising determining the presence or absence of the third target or its amplicon in the sample by:

- bringing the reaction mixture containing the sample or its derivative into contact with:

a third closed stem-loop oligonucleotide, wherein said third closed stem-loop oligonucleotide contains a double-stranded stem fragment of hybridized nucleotides connected to a loop fragment of a closed single-stranded oligonucleotide a consisting of unhybridized nucleotides, wherein:

the number of hybridized nucleotides and/or the sequence of hybridized nucleotides in the stem fragment of the third closed stem-loop oligonucleotide is different from the number and sequence of hybridized nucleotides in the stem fragments of the first and second closed stem-loop oligonucleotides;

the double-stranded stem fragment contains a fluorophore molecule linked to one strand and a quencher molecule linked to the opposite strand; and

enzymes capable of cleaving or destroying the single-stranded loop fragment of the third closed stem-loop oligonucleotide only when it is in contact with the target or its amplicon;

- treating the reaction mixture under conditions suitable to induce cleavage or destruction of the loop fragment of the third closed stem-loop oligonucleotide with enzymes, which allows to obtain a third open stem-loop oligonucleotide;

- detection of the presence or absence of the third oligonucleotide by processing the reaction mixture or its derivative when:

a third temperature at which the strands of the double-stranded stem fragment of the third disclosed stem-loop oligonucleotide dissociate, thereby facilitating the spatial separation of the fluorophore and quencher molecules of the stem fragment of the third disclosed stem-loop oligonucleotide and providing a third detectable signal,

and:

the third temperature is different from the first and second temperatures, and

detection of a signal at a third temperature indicates the presence of a third target in the sample, and failure to detect a signal at a third temperature indicates the absence of a third target in the sample.

Embodiment 26. The method of any one of Embodiments 1-24, further comprising determining the presence or absence of the third target or its amplicon in the sample by:

- bringing the reaction mixture containing the sample or its derivative into contact with:

a third closed stem-loop oligonucleotide, wherein said third closed stem-loop oligonucleotide contains a double-stranded stem fragment of hybridized nucleotides connected to a loop fragment of a closed single-stranded oligonucleotide a consisting of unhybridized nucleotides, wherein:

the number of hybridized nucleotides and/or the sequence of hybridized nucleotides in the stem fragment of the third closed stem-loop oligonucleotide matches the number and sequence of hybridized nucleotides in the stem fragments of the first or second closed stem-loop oligonucleotides;

the double-stranded stem fragment contains a fluorophore molecule connected to one strand and a quencher molecule connected to the opposite strand, wherein the fluorophore molecule connected to the third disclosed stem-loop oligonucleotide emits in a different region of the visible spectrum compared to the fluorophore of the first and/or or a second closed stem-loop oligonucleotide; and

enzymes capable of cleaving or destroying the single-stranded loop fragment of the third closed stem-loop oligonucleotide only when it is in contact with the target or its amplicon;

- treating the reaction mixture under conditions suitable to induce cleavage or destruction of the loop fragment of the third closed stem-loop oligonucleotide with enzymes, which allows to obtain a third open stem-loop oligonucleotide;

- detection of the presence or absence of the third oligonucleotide by processing the reaction mixture or its derivative when:

a third temperature at which the strands of the double-stranded stem fragment of the third disclosed stem-loop oligonucleotide dissociate, thereby facilitating the spatial separation of the fluorophore and quencher molecules of the stem fragment of the third disclosed stem-loop oligonucleotide and providing a third detectable signal,

and:

detection of a signal at a third temperature indicates the presence of a third target in the sample, and failure to detect a signal at a third temperature indicates the absence of a third target in the sample.

Embodiment 27. The method of Embodiment 26 wherein the third temperature is different from the first and/or second temperatures.

Embodiment 28. The method of any one of Embodiments 25-27, wherein said method comprises subjecting the reaction mixture to conditions suitable for any one or more of the following processes:

- binding of the third MNKzyme with the third target or its amplicon and hybridization of the substrate chains of the specified third MNKzyme with the loop fragment of the third closed stem-loop oligonucleotide, which facilitates the specified cleavage of the loop fragment of the third closed stem-loop oligonucleotide by the third MNKzyme with the formation of the third a disclosed stem-loop oligonucleotide;

- hybridization of the third target or its amplicon with the loop fragment of the third closed stem-loop oligonucleotide to form a double-stranded sequence for binding the third restriction endonuclease to associate with the second double-stranded sequence, which facilitates said cleavage of the loop fragment of the third closed stem-loop oligonucleotide "to form a third disclosed stem-loop oligonucleotide;

- hybridization of the third target or its amplicon with the loop fragment of the third closed stem-loop oligonucleotide to form a third double-stranded sequence containing the third target or its amplicon,

hybridization of the third primer-oligonucleotide with the third target or its amplicon with the formation of the third double-stranded sequence located above (in the 5' direction) relative to the third double-stranded sequence containing the third target or its amplicon, association of the third enzyme with exonuclease activity with the loop fragment of the third closed a stem-loop oligonucleotide at or near the end of the third oligonucleotide primer, and

catalytic activity of a third enzyme having exonuclease activity, which facilitates the destruction of the loop fragment of the third double-stranded sequence containing the third target or amplicon, and the formation of the third open stem-loop oligonucleotide;

- hybridization of the third target or its amplicon with the loop fragment of the third closed stem-loop oligonucleotide to form a third double-stranded sequence containing the third target or its amplicon,

association of the third enzyme having exonuclease activity with the third double strand sequence containing the third target or its amplicon and the catalytic activity of the third enzyme having exonuclease activity, which facilitates the destruction of the loop fragment of the third double strand sequence containing the third target or amplicon and the formation of the third opened oligonucleotide stem-loop type;

- binding of the third cofactor to the DNAzyme and/or binding of the third cofactor to the ribozyme, which imparts catalytic activity to the DNAzyme and/or ribozyme,

hybridization of the DNAzyme and/or ribozyme with the loop fragment of the third closed stem-loop oligonucleotide,

catalytic activity of the DNAzyme and/or ribozyme to facilitate cleavage of the loop fragment of the third closed stem-loop oligonucleotide and the formation of the third open stem-loop oligonucleotide.

and:

the third target is the third cofactor.

Embodiment 29. The method of Embodiment 28 wherein the cofactor is a metal ion (eg, Mg ²⁺ , Mn ²⁺ , Ca ²⁺ , Pb ²⁺ ).

Embodiment 30. The method of Embodiment 28, wherein the third restriction endonuclease is a nicking endonuclease capable of associating with and cleaving the loop strand of said double-stranded sequence containing the third target or its amplicon.

Embodiment 31. The method of Embodiment 28 wherein the third target is a nucleotide sequence or amplicon thereof capable of hybridizing to the sensor strands of the third MNKzyme, thereby facilitating assembly of the third MNKzyme.

Embodiment 32. The method of Embodiment 28, wherein:

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

- enzymes include enzymes with an aptamer capable of binding to a third target; and

- binding of the third target to the aptamer is capable of making enzymes with the aptamer catalytically active.

Embodiment 33. The method of Embodiment 32 wherein the aptamer enzymes comprise any one or more of the following classes of compounds: apta-DNAzymes, apta-ribozymes, apta-MNKzymes.

Embodiment 34. The method of Embodiment 28, wherein:

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

- the reaction mixture additionally contains an oligonucleotide sequence capable of hybridizing with the sensor chains of the third MNKzyme, thereby facilitating the assembly of the third MNKzyme;

the third MNKzyme contains an aptamer sequence capable of binding to a third target; and

binding of the third target to the aptamer sequence of the third MNKzyme is capable of making the third MNKzyme catalytically active, facilitating the removal of the inhibitory molecule associated with the aptamer of the third MNKzyme.

Embodiment 35. A method according to any one of embodiments 28 or 30-34, wherein:

the reaction mixture contains said third MNKzyme and one or both of said first and second MNKzymes; and

- the sequence of the loop fragment of the third closed stem-loop oligonucleotide capable of hybridizing with the substrate chains of the third MNKzyme differs from:

sequences of the loop fragment of the first closed stem-loop oligonucleotide capable of hybridizing with the substrate chains of the first MNKzyme, and/or

sequences of the loop fragment of the second closed stem-loop oligonucleotide capable of hybridizing with the substrate chains of the first MNCzyme.

Embodiment 36. The method of any of embodiments 25 or 28-35, wherein:

- the number of hybridized nucleotides and/or the sequence of hybridized nucleotides in the stem fragment of the third closed stem-loop oligonucleotide differs from the number and sequence of hybridized nucleotides in the stem fragments of the first and second closed stem-loop oligonucleotides;

- the third temperature is different from the first and second temperatures; and

- the fluorophore of the third closed and open stem-loop oligonucleotide emits in the same color region of the visible spectrum as the fluorophores of the first and second closed and open stem-loop oligonucleotides.

Embodiment 37. The method of any of embodiments 25 or 28-36 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 ".

Embodiment 38. The method of Embodiment 37 wherein the fluorophore of the third closed and open stem-loop oligonucleotides and the fluorophore of the first closed and opened oligonucleotides and/or the fluorophore of the second closed and opened oligonucleotides are detected on the same fluorescence channel of the device .

Embodiment 39. The method of any of embodiments 25-35 or 36, wherein:

- the fluorophore of the first closed and open stem-loop oligonucleotide emits in the same color region of the visible spectrum as the fluorophore of the second closed and open stem-loop oligonucleotide; and

- the fluorophore of the third closed and open stem-loop oligonucleotide emits in the color region of the visible spectrum, which differs from the emission region of the fluorophore of the first and second closed and open stem-loop oligonucleotides.

Embodiment 40. The method of any one of Embodiments 25-39 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°С, 7°С, 8°С, 9°С, 10°С, 11°С, 12°С, 13°С, 14°С, 15°С, 16°С, 17°С, 18 °С, 19°С, 20°С, 25°С, 30°С, 35°С, 40°С, 45°С, 50°С, 55°С, 60°С, 65°С, 70°С , 75°С, 80°С.

Embodiment 41. The method of any one of Embodiments 1-40 wherein melt curve analysis is used for any specified signal detection or any specified signal failure.

Embodiment 42. The method of any 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°С, 8°С, 9°С, 10°С, 11°С, 12°С, 13°С, 14°С, 15°С, 16°С, 17°С, 18°С, 19°С С, 20°С, 25°С, 30°С, 35°С, 40°С, 45°С, 50°С, 55°С, 60°С, 65°С, 70°С, 75°С, 80°C.

Embodiment 43. The method of any one of Embodiments 1 to 42, wherein any said amplicon is produced by any of polymerase chain reaction (PCR), strand displacement amplification (SDA), helicase dependent amplification (HDA), recombinase polymerase amplification (RPA), loop isothermal amplification (LAMP), rolling ring amplification (RCA), transcription-mediated amplification (TMA), self-sustaining sequence replication (3SR), nucleic acid sequence-based amplification (NASBA), or polymerase chain reaction with reverse transcription (RT-PCR).

Embodiment 44. The method of any one of Embodiments 1-43, wherein the sample is a biological sample obtained from a subject.

Embodiment 45. The method of any one of Embodiments 1-43, wherein the method is performed in vitro.

Embodiment 46. The method of any one of Embodiments 1-43, wherein the method is performed ex vivo.

Embodiment 47. A composition comprising:

first and second closed stem-loop oligonucleotides, wherein each of the closed stem-loop oligonucleotides contains a double-stranded stem fragment formed by hybridized nucleotides and connected to a single-stranded loop fragment formed by unhybridized nucleotides, wherein:

the number of hybridized nucleotides and/or the sequence of hybridized nucleotides in the stem fragment of the first and second closed stem-loop oligonucleotides are different, and

each of the double-stranded stem fragments contains a fluorophore molecule attached to one strand and a quencher molecule attached to the opposite strand,

moreover, the fluorophore molecules emit in the same color region of the visible spectrum, and the melting temperature (Tm) of the double-stranded stem fragment of the first closed stem-loop oligonucleotide differs from the Tm of the double-stranded stem fragment of the second closed stem-loop oligonucleotide.

Embodiment 48. The composition of Embodiment 47 wherein the single stranded loop fragment of the first closed stem-loop oligonucleotide differs from the sequence of the single stranded loop fragment of the second closed stem-loop oligonucleotide.

Embodiment 49. The composition of Embodiment 47 or 48, further comprising:

a first MNKzyme containing substrate chains capable of hybridizing to a closed single-stranded loop fragment of a first closed stem-loop oligonucleotide; and

a second MNKzyme containing substrate chains capable of hybridizing to a single-stranded loop fragment of a second closed stem-loop oligonucleotide.

Embodiment 50. The composition of Embodiment 49, wherein:

substrate chains of the first MNCzyme hybridize with a single-stranded loop fragment of the first closed stem-loop oligonucleotide; and

the substrate chains of the second MNCzyme hybridize with a single-stranded loop fragment of the second closed stem-loop oligonucleotide.

Embodiment 51. The composition of Embodiment 50, wherein:

the first and/or second MNKzymes contain an aptamer sequence associated with the analyzed target compound, protein, compound or molecule, and sensor strands hybridized to the oligonucleotide sequence; and

the first MNKzyme is designed to detect a target that is different from the target of the second MNKzyme.

Embodiment 52. The composition of Embodiment 50, wherein:

the first and/or second MNKzymes contain sensory strands that hybridize to the target sequence, and the first MNKzyme is designed to detect a target that is different from the target of the second MNKzyme.

Embodiment 53. The composition of any one of Embodiments 47-52, further comprising:

a first oligonucleotide sequence hybridizing to said single-stranded loop fragment of a first closed stem-loop oligonucleotide that differs from the sequence of a single-stranded loop fragment of a second closed stem-loop oligonucleotide, thereby forming a double-stranded sequence comprising said first oligonucleotide;

a second oligonucleotide sequence hybridizing to said single-stranded loop fragment of a second closed stem-loop oligonucleotide different from the sequence of the single-stranded loop fragment of the first closed stem-loop oligonucleotide, thereby forming a double-stranded sequence comprising said second oligonucleotide;

a restriction endonuclease associated with a double-stranded sequence comprising said first oligonucleotide and capable of cleaving it; and/or

a restriction endonuclease associated with a double-stranded sequence comprising said second oligonucleotide and capable of cleaving it.

Embodiment 54. The composition of any one of Embodiments 47-53, further comprising:

a first double-stranded sequence comprising a first target oligonucleotide sequence hybridizing to said single-stranded loop fragment of the first closed stem-loop oligonucleotide different from the sequence of the single-stranded loop fragment of the second closed stem-loop oligonucleotide, thereby forming the first double-stranded sequence, containing the specified first oligonucleotide target;

a second double-stranded sequence comprising a second target oligonucleotide sequence hybridizing to said single-stranded loop fragment of the second closed stem-loop oligonucleotide different from the sequence of the single-stranded loop fragment of the first closed stem-loop oligonucleotide, thereby forming a double-stranded sequence containing said second target oligonucleotide;

a first primer oligonucleotide hybridizing to the first target oligonucleotide upstream (in the 5' direction) of said first double-stranded sequence, and a first enzyme having exonuclease activity (e.g., polymerase, exonuclease) associated with the loop fragment of the first closed stem-type oligonucleotide "loop" at or near the end of the first oligonucleotide primer; and/or

a second primer oligonucleotide hybridizing to a second target oligonucleotide upstream (in the 5' direction) of said second double-stranded sequence, and a second enzyme having exonuclease activity (e.g., polymerase, exonuclease) associated with the loop fragment of the second closed stem-type oligonucleotide -loop" at the end of the second oligonucleotide primer

Embodiment 55. The composition of any one of Embodiments 47-54, further comprising:

a first double-stranded sequence comprising a first target oligonucleotide sequence hybridizing to said single-stranded loop fragment of the first closed stem-loop oligonucleotide different from the sequence of the single-stranded loop fragment of the second closed stem-loop oligonucleotide, thereby forming the first double-stranded sequence, containing the specified first oligonucleotide target;

a second double-stranded sequence comprising a second target oligonucleotide sequence hybridizing to said single-stranded loop fragment of the second closed stem-loop oligonucleotide different from the sequence of the single-stranded loop fragment of the second closed stem-loop oligonucleotide, thereby forming a double-stranded sequence containing said second target oligonucleotide;

wherein the first enzyme having exonuclease activity is associated with the first double stranded sequence and the second enzyme having exonuclease activity is associated with the second double stranded sequence.

Embodiment 56. The composition of any one of Embodiments 47-55, further comprising:

the first DNAzyme and/or the first ribozyme hybridizing to a loop fragment of the first closed stem-loop oligonucleotide that differs from the sequence of the single-stranded loop fragment of the second closed stem-loop oligonucleotide,

a second DNAzyme and/or a second ribozyme hybridizing to a loop fragment of the second closed stem-loop oligonucleotide that differs from the sequence of the single-stranded loop fragment of the first closed stem-loop oligonucleotide,

cofactors for each said DNAzyme capable of conferring catalytic activity on each said DNAzyme and thus inducing the cleaving activity of any said DNAzyme hybridized to the loop fragment.

cofactors for each said ribozyme capable of conferring catalytic activity on each said ribozyme and thus inducing the cleavage activity of any said loop hybridized ribozyme.

Embodiment 57. The composition of any one of Embodiments 47-56, further comprising:

third closed stem-loop oligonucleotides, wherein said third closed stem-loop oligonucleotides comprise a double-stranded stem fragment of hybridized nucleotides connected to a single-stranded loop fragment of unhybridized nucleotides, wherein:

the number of hybridized nucleotides and/or the sequence of hybridized nucleotides in the stem fragment of the third closed stem-loop oligonucleotides differs from the number and sequence of hybridized nucleotides in the stem fragments of the first and second closed stem-loop oligonucleotides, and

the double-stranded stem fragment of the third closed stem-loop oligonucleotides contains a fluorophore molecule connected to one strand and a quencher molecule connected to the opposite strand, and the fluorophore molecule of the third closed stem-loop oligonucleotide emits in a different region of the visible spectrum compared to with fluorophore molecules of the first and second closed stem-loop oligonucleotides.

Embodiment 58. The composition of any one of Embodiments 47-56, further comprising:

third closed stem-loop oligonucleotides, wherein said third closed stem-loop oligonucleotides comprise a double-stranded stem fragment of hybridized nucleotides connected to a single-stranded loop fragment of unhybridized nucleotides, wherein:

the number of hybridized nucleotides and/or the sequence of hybridized nucleotides in the stem fragment of the third closed stem-loop oligonucleotides differs from the number and sequence of hybridized nucleotides in the stem fragments of the first and second closed stem-loop oligonucleotides, and

the double-stranded stem fragment of the third closed stem-loop oligonucleotides contains a fluorophore molecule connected to one strand and a quencher molecule connected to the opposite strand, and the fluorophore molecule of the third closed stem-loop oligonucleotide emits in the same region of the visible spectrum along compared with the fluorophore molecules of the first and second closed stem-loop oligonucleotides.

Embodiment 59. The composition of any one of Embodiments 47-56, further comprising:

third closed stem-loop oligonucleotides, wherein said third closed stem-loop oligonucleotides comprise a double-stranded stem fragment of hybridized nucleotides connected to a single-stranded loop fragment of unhybridized nucleotides, wherein:

the number of hybridized nucleotides and/or the sequence of hybridized nucleotides in the stem fragment of the third closed stem-loop oligonucleotides matches the number and sequence of hybridized nucleotides in the stem fragments of the first or second closed stem-loop oligonucleotides, and

the double-stranded stem fragment of the third closed stem-loop oligonucleotides contains a fluorophore molecule connected to one strand and a quencher molecule connected to the opposite strand, and the fluorophore molecule of the third closed stem-loop oligonucleotide emits in a different region of the visible spectrum compared to with fluorophore molecules of the first and second closed stem-loop oligonucleotides.

The present invention also relates to Embodiments 1-81 listed below:

Embodiment 1. A method for determining the presence or absence of first and second targets in a sample, said method comprising:

(a) obtaining a reaction mixture by bringing the sample or its derivative, presumably containing the first and/or second targets or their amplicons, into contact with:

the first and second closed stem-loop oligonucleotides, each of the closed stem-loop oligonucleotides containing a double-stranded stem fragment formed by hybridized nucleotides and connected to a closed single-stranded loop fragment formed by unhybridized nucleotides, wherein:

the number of hybridized nucleotides and/or the sequence of hybridized nucleotides in the stem fragment of the first and second closed stem-loop oligonucleotides are different, and

enzymes capable of cleaving or destroying single-stranded loop fragments of the first and second closed stem-loop oligonucleotides only upon contact with the target or its amplicon;

(b) working up the reaction mixture:

- under conditions suitable for the enzymes to induce cleavage or destruction of the loop fragment of the first and second closed stem-loop oligonucleotides, thereby forming the first and second open stem-loop oligonucleotides;

at or above the first temperature at which the double-stranded stem fragment chains of the first disclosed stem-loop oligonucleotide dissociate, thereby facilitating the spatial separation of the fluorophore and quencher molecules of the stem fragment of the first disclosed stem-loop oligonucleotide and providing the first detectable signal, and

at or above a second temperature at which the double-stranded stem fragment chains of the second disclosed stem-loop oligonucleotide dissociate, thereby facilitating the spatial separation of the fluorophore and quencher molecules of the stem fragment of the second disclosed stem-loop oligonucleotide and providing a second detectable fluorescent signal;

and:

the first temperature is lower than the second temperature, and

the fluorophore of the first disclosed stem-loop oligonucleotide and the fluorophore of the second disclosed stem-loop oligonucleotide emit in the same color region of the visible spectrum; and

(c) detecting levels of said first and second fluorescent signals at one or more temperatures, including a temperature equal to or greater than the second temperature, which allows the presence or absence of said targets to be determined in the sample.

Embodiment 2. The method of Embodiment 1 wherein the enzymes contain multicomponent nucleozymes (MNCzymes) and said treatment of the reaction mixture comprises treating the reaction mixture under conditions suitable for:

binding a first multicomponent nucleo- zyme (MNKzyme) to a first target or amplicon thereof; and hybridizing the substrate strands of said first MNKzyme with a loop fragment of the first closed stem-loop oligonucleotide, thereby facilitating said cleavage of the loop fragment of the first closed stem-loop oligonucleotide by the first MNKzyme to form the first open stem-loop oligonucleotide.

Embodiment 3. The method of Embodiment 2 wherein the first target is a nucleotide sequence or amplicon thereof capable of hybridizing to the sensor strands of the first MNKzyme, thereby facilitating assembly of the first MNKzyme.

Embodiment 4. The method of Embodiment 1, wherein:

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

- enzymes include enzymes with an aptamer capable of binding to the first target; and

binding of the first target to the aptamer is capable of making enzymes with the aptamer catalytically active.

Embodiment 5. The method of Embodiment 4 wherein the aptamer enzymes comprise any one or more of the following classes of compounds: apta-DNAzymes, apta-ribozymes, apta-MNAzymes.

Embodiment 6. A method according to any one of embodiments 2, 4, or 5, wherein:

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

- the reaction mixture additionally contains an oligonucleotide sequence capable of hybridizing with the sensor chains of the first MNKzyme, thereby facilitating the assembly of the first MNKzyme;

the first MNKzyme contains an aptamer sequence capable of binding to the first target; and

- binding of the target to the aptamer can make the first MNKzyme catalytically active.

Embodiment 7. The method of any one of embodiments 1-6, wherein said closed stem-loop oligonucleotides do not hybridize to said target or amplicon thereof during said cleavage or degradation by enzymes.

Embodiment 8. The method of any one of Embodiments 1-6 wherein the enzymes include restriction endonucleases and said treatment of the reaction mixture comprises:

processing the reaction mixture under conditions suitable for hybridizing the first target or its amplicon with the loop fragment of the first closed stem-loop oligonucleotide to form a double-stranded sequence for binding the first restriction endonuclease, which facilitates said cleavage of the loop fragment of the first closed stem-loop oligonucleotide to form the first disclosed stem-loop oligonucleotide.

Embodiment 9. The method of Embodiment 8, wherein the restriction endonuclease is a nicking endonuclease capable of binding to and cleaving the loop strand of said double-stranded sequence for the first restriction endonuclease.

Embodiment 10. The method of any one of Embodiments 1 to 6, wherein the enzymes have exonuclease activity (e.g., polymerase enzymes, exonucleases) and said treatment of the reaction mixture comprises:

processing the reaction mixture under conditions suitable for:

- hybridization of the first target or its amplicon with the loop fragment of the first closed stem-loop oligonucleotide to form the first double-stranded sequence containing the first target or its amplicon,

- hybridization of the first primer-oligonucleotide with the first target or its amplicon with the formation of the second double-stranded sequence located upstream (in the direction of 5') relative to the first double-stranded sequence containing the first target or its amplicon

association of the first enzyme having exonuclease activity with the loop fragment of the first closed stem-loop oligonucleotide at or near the end of the first primer-oligonucleotide, and

- catalytic activity of the first enzyme having exonuclease activity, which facilitates the destruction of the loop fragment of the first double-stranded sequence containing the first target or amplicon, and the formation of the first open stem-loop oligonucleotide.

Embodiment 11. The method of any one of Embodiments 1-6 wherein the enzymes have exonuclease activity and said treatment of the reaction mixture comprises:

processing the reaction mixture under conditions suitable for:

- hybridization of the first target or its amplicon with the loop fragment of the first closed stem-loop oligonucleotide to form the first double-stranded sequence containing the first target or its amplicon,

association of the first enzyme having exonuclease activity with the first double-stranded sequence containing the first target or its amplicon, and

- catalytic activity of the first enzyme having exonuclease activity, which facilitates the destruction of the loop fragment of the first double-stranded sequence containing the first target or amplicon, and the formation of the first open stem-loop oligonucleotide.

Embodiment 12. The method of any one of Embodiments 1-11, wherein the enzymes comprise DNAzymes and/or ribozymes requiring a first cofactor to exhibit catalytic activity, and said treatment of the reaction mixture comprises treating the reaction mixture under conditions suitable for:

- linking said first cofactor to a DNAzyme and/or linking said first cofactor to a ribozyme conferring catalytic activity on the DNAzyme and/or ribozyme,

- hybridization of the DNAzyme and/or ribozyme with the loop fragment of the first closed stem-loop oligonucleotide,

- catalytic activity of the DNAzyme and/or ribozyme to facilitate cleavage of the loop fragment of the first closed stem-loop oligonucleotide and the formation of the first open stem-loop oligonucleotide.

and:

the first target is the first cofactor.

Embodiment 13. The method of Embodiment 12 wherein the first cofactor is a metal ion (eg, Mg ²⁺ , Mn ²⁺ , Ca ²⁺ , Pb ²⁺ ).

Embodiment 14. The method of any one of Embodiments 1-13, wherein said treatment further comprises treating the reaction mixture under conditions suitable for any one or more of the following processes:

- binding of the second MNKzyme with the second target or its amplicon and hybridization of the substrate chains of the specified second MNKzyme with the loop fragment of the second closed stem-loop oligonucleotide, which facilitates the specified cleavage of the loop fragment of the second closed stem-loop oligonucleotide by the second MNKzyme with the formation of the second a disclosed stem-loop oligonucleotide;

- hybridization of the second target or its amplicon with the loop fragment of the second closed stem-loop oligonucleotide to form a double-stranded sequence for binding the second restriction endonuclease to associate with the second double-stranded sequence, which facilitates said cleavage of the loop fragment of the second closed stem-loop oligonucleotide " to form a second open stem-loop oligonucleotide,

- hybridization of the second target or its amplicon with the loop fragment of the second closed stem-loop oligonucleotide to form a second double-stranded sequence containing the second target or its amplicon,

hybridization of the second primer-oligonucleotide with the second target or its amplicon to form a second double-stranded sequence located upstream (in the 5' direction) relative to the second double-stranded sequence containing the second target or its amplicon,

association of the second enzyme having exonuclease activity with the loop fragment of the second closed stem-loop oligonucleotide at or near the end of the second primer-oligonucleotide, and

catalytic activity of a second enzyme having exonuclease activity, which facilitates the destruction of the loop fragment of the second double-stranded sequence containing the second target or amplicon, and the formation of the second open stem-loop oligonucleotide;

- hybridization of the second target or its amplicon with the loop fragment of the second closed stem-loop oligonucleotide to form a second double-stranded sequence containing the second target or its amplicon,

association of a second enzyme having exonuclease activity with a second double-stranded sequence containing a second target or its amplicon, and

catalytic activity of a second enzyme having exonuclease activity, which facilitates the destruction of the loop fragment of the second double-stranded sequence containing the second target or amplicon, and the formation of the second open stem-loop oligonucleotide;

- binding of the second cofactor to the DNAzyme and/or binding of the second cofactor to the ribozyme conferring catalytic activity on the DNAzyme and/or ribozyme,

hybridization of the DNAzyme and/or ribozyme with the loop fragment of the second closed stem-loop oligonucleotide,

catalytic activity of the DNAzyme and/or ribozyme to facilitate cleavage of the loop fragment of the second closed stem-loop oligonucleotide and the formation of the second open stem-loop oligonucleotide.

and:

the second target is the second cofactor.

Embodiment 15. The method of Embodiment 14 wherein the second cofactor is a metal ion (eg, Mg ²⁺ , Mn ²⁺ , Ca ²⁺ , Pb ²⁺ ).

Embodiment 16. The method of Embodiment 14 wherein the second restriction endonuclease is a nicking endonuclease capable of associating with and cleaving the loop strand of said double-stranded sequence containing the second target or its amplicon.

Embodiment 17. The method of Embodiment 16 wherein the first and second restriction endonucleases are different types of restriction endonucleases.

Embodiment 18. The method of Embodiment 16 wherein the first and second restriction endonucleases are the same type of restriction endonucleases.

Embodiment 19. The method of Embodiment 14 wherein the second target is a nucleotide sequence or amplicon thereof capable of hybridizing to the sensor strands of the second MNKzyme, thereby facilitating assembly of the second MNKzyme.

Embodiment 20. The method of Embodiment 14, wherein:

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

- enzymes include enzymes with an aptamer capable of binding to a second target; and

binding of the second target to the aptamer is capable of making enzymes with the aptamer catalytically active.

Embodiment 21. The method of Embodiment 20 wherein the aptamer enzymes comprise any one or more of the following classes of compounds: apta-DNAzymes, apta-ribozymes, apta-MNKzymes.

Embodiment 22. The method of Embodiment 14, 20, or 21, wherein:

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

- the reaction mixture additionally contains an oligonucleotide sequence capable of hybridizing with the sensor chains of the second MNKzyme, thereby facilitating the assembly of the second MNKzyme;

the second MNKzyme contains an aptamer sequence capable of binding to the second target; and

- binding of the second target to the aptamer sequence of the second MNKzyme is able to make the second MNKzyme catalytically active, facilitating the removal of the inhibitory molecule associated with the aptamer of the second MNKzyme.

Embodiment 23. The method of any one of Embodiments 1-22, wherein the fluorophore of the first closed and opened stem-loop oligonucleotides is the same as the fluorophore of the second closed and opened stem-loop oligonucleotides.

Embodiment 24. The method of any one of embodiments 14-23, wherein:

- the reaction mixture contains the specified first and second MNCzyme; and

- the sequence of the loop fragment of the first closed stem-loop oligonucleotide capable of hybridizing with the substrate chains of the first MNCzyme differs from the sequence of the loop fragment of the second closed stem-loop oligonucleotide capable of hybridizing with the substrate chains of the second MNCzyme.

Embodiment 25. The method of any one of embodiments 1-24, wherein the fluorophore of the first closed and open stem-loop oligonucleotides and the fluorophore of the second closed and opened stem-loop oligonucleotides are detected on the same fluorescent emission channel of the device.

Embodiment 26. The method of any of embodiments 1-25, wherein said enzymes do not induce cleavage or degradation of any of said target or its amplicon.

Embodiment 27. The method of any one of Embodiments 1-26, wherein the first and second closed stem-loop oligonucleotides each consist of said double-stranded stem fragment and said single-stranded loop fragment.

Embodiment 28. The method of any one of Embodiments 1-27, further comprising determining the presence or absence of the third target or its amplicon in the sample by:

(d) bringing the reaction mixture containing the sample or its derivative into contact with:

a third closed stem-loop oligonucleotide, wherein said third closed stem-loop oligonucleotide contains a double-stranded stem fragment of hybridized nucleotides connected to a loop fragment of a closed single-stranded oligonucleotide consisting of unhybridized nucleotides, wherein:

the number of hybridized nucleotides and/or the sequence of hybridized nucleotides in the stem fragment of the third closed stem-loop oligonucleotide is different from the number and sequence of hybridized nucleotides in the stem fragments of the first and second closed stem-loop oligonucleotides;

the double-stranded stem fragment contains a fluorophore molecule linked to one strand and a quencher molecule linked to the opposite strand; and

enzymes capable of cleaving or destroying the single-stranded loop fragment of the third closed stem-loop oligonucleotide only when it is in contact with the target or its amplicon;

(e) working up the reaction mixture:

under conditions suitable to induce cleavage or disruption of the loop fragment of the third closed stem-loop oligonucleotide by enzymes, thereby producing a third open stem-loop oligonucleotide;

at a third temperature at or above which the strands of the double-stranded stem fragment of the third disclosed stem-loop oligonucleotide dissociate, thereby facilitating the spatial separation of the fluorophore and quencher molecules of the stem fragment of the third disclosed stem-loop oligonucleotide and producing a third detectable fluorescent signal; and:

the third temperature is higher than the first and second temperatures, and

(f) detecting levels of said first, second, and third fluorescent signals at one or more temperatures, including a temperature equal to or greater than the third temperature, to determine the presence or absence of said targets in the sample.

Embodiment 29. The method of any one of Embodiments 1-27, further comprising determining the presence or absence of the third target or its amplicon in the sample by:

(d) bringing the reaction mixture containing the sample or its derivative into contact with:

a third closed stem-loop oligonucleotide, wherein said third closed stem-loop oligonucleotide contains a double-stranded stem fragment of hybridized nucleotides connected to a loop fragment of a closed single-stranded oligonucleotide consisting of unhybridized nucleotides, wherein:

the number of hybridized nucleotides and/or the sequence of hybridized nucleotides in the stem fragment of the third closed stem-loop oligonucleotide matches the number and sequence of hybridized nucleotides in the stem fragments of the first or second closed stem-loop oligonucleotides;

the double-stranded stem fragment contains a fluorophore molecule connected to one strand and a quencher molecule connected to the opposite strand, and the fluorophore molecule connected to the third disclosed stem-loop oligonucleotide emits in a different region of the visible spectrum compared to the fluorophore of the first and/or or a second closed stem-loop oligonucleotide; and

enzymes capable of cleaving or destroying the single-stranded loop fragment of the third closed stem-loop oligonucleotide only when it is in contact with the target or its amplicon;

(e) working up the reaction mixture:

- under conditions suitable for inducing cleavage or destruction of the loop fragment of the third closed stem-loop oligonucleotide by enzymes, which allows to obtain a third open stem-loop oligonucleotide;

at a third temperature at which the double-stranded stem fragment of the third disclosed stem-loop oligonucleotide dissociates, thereby facilitating the spatial separation of the fluorophore and quencher molecules of the stem fragment of the third disclosed stem-loop oligonucleotide and providing a third detectable fluorescent signal;

(f) detecting levels of said first, second, and third fluorescent signals at one or more temperatures, including a temperature equal to or greater than the third temperature, to determine the presence or absence of said targets in the sample.

Embodiment 30. The method of Embodiment 29 wherein the third temperature is different from the first and/or second temperatures.

Embodiment 31. The method of any one of Embodiments 28-30, wherein said method comprises subjecting the reaction mixture to conditions suitable for any one or more of the following processes:

binding the third MNKzyme to a third target or its amplicon and hybridization of the substrate chains of said third MNKzyme with the loop fragment of the third closed stem-loop oligonucleotide, which facilitates said cleavage of the loop fragment of the third closed stem-loop oligonucleotide by the third MNKzyme to form the third a disclosed stem-loop oligonucleotide;

- hybridization of the third target or its amplicon with the loop fragment of the third closed stem-loop oligonucleotide to form a double-stranded sequence for binding the third restriction endonuclease to associate with the second double-stranded sequence, which facilitates said cleavage of the loop fragment of the third closed stem-loop oligonucleotide "to form a third disclosed stem-loop oligonucleotide;

- hybridization of the third target or its amplicon with the loop fragment of the third closed stem-loop oligonucleotide to form a third double-stranded sequence containing the third target or its amplicon,

hybridization of the third primer-oligonucleotide with the third target or its amplicon with the formation of the third double-stranded sequence located above (in the 5' direction) relative to the third double-stranded sequence containing the third target or its amplicon, association of the third enzyme with exonuclease activity with the loop fragment of the third closed a stem-loop oligonucleotide at or near the end of the third oligonucleotide primer, and

catalytic activity of a third enzyme having exonuclease activity, which facilitates the destruction of the loop fragment of the third double-stranded sequence containing the third target or amplicon, and the formation of the third open stem-loop oligonucleotide;

- hybridization of the third target or its amplicon with the loop fragment of the third closed stem-loop oligonucleotide to form a third double-stranded sequence containing the third target or its amplicon,

association of a third enzyme having exonuclease activity with a third double-stranded sequence containing a third target or its amplicon, and

catalytic activity of a third enzyme having exonuclease activity, which facilitates the destruction of the loop fragment of the third double-stranded sequence containing the third target or amplicon, and the formation of the third open stem-loop oligonucleotide;

- binding of the third cofactor to the DNAzyme and/or binding of the third cofactor to the ribozyme, which imparts catalytic activity to the DNAzyme and/or ribozyme,

hybridization of the DNAzyme and/or ribozyme with the loop fragment of the third closed stem-loop oligonucleotide,

catalytic activity of the DNAzyme and/or ribozyme to facilitate cleavage of the loop fragment of the third closed stem-loop oligonucleotide and the formation of the third open stem-loop oligonucleotide.

and:

the third target is the third cofactor.

Embodiment 32. The method of Embodiment 31 wherein the cofactor is a metal ion (eg, Mg ²⁺ , Mn ²⁺ , Ca ²⁺ , Pb ²⁺ ).

Embodiment 33. The method of Embodiment 31, wherein the third restriction endonuclease is a nicking endonuclease capable of associating with and cleaving the loop strand of said double-stranded sequence containing the third target or its amplicon.

Embodiment 34. The method of Embodiment 31 wherein the third target is a nucleotide sequence or amplicon thereof capable of hybridizing to the sensor strands of the third MNKzyme, thereby facilitating assembly of the third MNKzyme.

Embodiment 35. The method of Embodiment 31, wherein:

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

- enzymes include enzymes with an aptamer capable of binding to a third target; and

- binding of the third target to the aptamer is capable of making enzymes with the aptamer catalytically active.

Embodiment 36. The method of Embodiment 35 wherein the aptamer enzymes comprise any one or more of the following classes of compounds: apta-DNAzymes, apta-ribozymes, apta-MNKzymes.

Embodiment 37. The method of Embodiment 31, wherein:

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

- the reaction mixture additionally contains an oligonucleotide sequence capable of hybridizing with the sensor chains of the third MNKzyme, thereby facilitating the assembly of the third MNKzyme;

the third MNKzyme contains an aptamer sequence capable of binding to a third target; and

binding of the third target to the aptamer sequence of the third MNKzyme is capable of making the third MNKzyme catalytically active, facilitating the removal of the inhibitory molecule associated with the aptamer of the third MNKzyme.

Embodiment 38. The method of any of embodiments 31 or 33-37, wherein:

the reaction mixture contains said third MNKzyme and one or both of said first and second MNKzymes; and

- the sequence of the loop fragment of the third closed stem-loop oligonucleotide capable of hybridizing with the substrate chains of the third MNKzyme differs from:

sequences of the loop fragment of the first closed stem-loop oligonucleotide capable of hybridizing with the substrate chains of the first MNKzyme, and/or

sequences of the loop fragment of the second closed stem-loop oligonucleotide capable of hybridizing with the substrate chains of the first MNCzyme.

Embodiment 39. The method of any of embodiments 28 or 31-38, wherein:

- the number of hybridized nucleotides and/or the sequence of hybridized nucleotides in the stem fragment of the third closed stem-loop oligonucleotide differs from the number and sequence of hybridized nucleotides in the stem fragments of the first and second closed stem-loop oligonucleotides;

- the third temperature is different from the first and second temperatures; and

- the fluorophore of the third closed and open stem-loop oligonucleotide emits in the same color region of the visible spectrum as the fluorophores of the first and second closed and open stem-loop oligonucleotides.

Embodiment 40. The method of any of embodiments 28 or 31-39 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 ".

Embodiment 41. The method of Embodiment 40, wherein the fluorophore of the third closed and opened stem-loop oligonucleotides and the fluorophore of the first closed and opened oligonucleotides and/or the fluorophore of the second closed and opened stem-loop oligonucleotides are detected on one and the same fluorescence emission channel of the device.

Embodiment 42. The method of any of embodiments 28-38 or 39, wherein:

- the fluorophore of the first closed and open stem-loop oligonucleotide emits in the same color region of the visible spectrum as the fluorophore of the second closed and open stem-loop oligonucleotide; and

- the fluorophore of the third closed and open stem-loop oligonucleotide emits in the color region of the visible spectrum, which differs from the emission region of the fluorophore of the first and second closed and open stem-loop oligonucleotides.

Embodiment 43. The method of any of embodiments 28-42 wherein the first 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°С, 7°С, 8°С, 9°С, 10°С, 11°С, 12°С, 13°С, 14°С, 15°С, 16°С, 17°С, 18 °С, 19°С, 20°С, 25°С, 30°С, 35°С, 40°С, 45°С, 50°С, 55°С, 60°С, 65°С, 70°С , 75°С, 80°С.

Embodiment 44. The method of any of embodiments 28-43, wherein detection of a third fluorescent signal at a third temperature indicates the presence of a third target in the sample, and failure to detect a third fluorescent signal at the third temperature indicates the absence of a third target in the sample.

Embodiment 45. The method of any one of Embodiments 1-44, wherein:

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

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

Embodiment 46. The method of any one of Embodiments 1-44, wherein said determining the presence or absence of first and second targets comprises analyzing a melt curve using said first and second fluorescent signals.

Embodiment 47. The method of any of embodiments 1-44, wherein part (c) comprises detecting levels of said first and second fluorescent signals when:

- a temperature equal to or greater than the second temperature; and

- a temperature equal to or greater than the first temperature and not reaching the second temperature.

Embodiment 48. The method of Embodiment 47, wherein part (c) further comprises detecting levels of said first and second fluorescent signals at a temperature less than the second temperature.

Embodiment 49. The method of Embodiment 47 or Embodiment 48, wherein part (c) comprises detecting levels of said first and second fluorescent signals during and/or after completion of a nucleic acid amplification reaction.

Embodiment 50. The method of any one of Embodiments 47-49, 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.

Embodiment 51. The method of any one of embodiments 47-50, further comprising obtaining a positive control fluorescent signal of the first target by repeating said method on a separate control sample containing said first target.

Embodiment 52. The method of Embodiment 51 wherein the control sample containing the first target contains the first target at a known concentration.

Embodiment 53. The method of Embodiment 51 or Embodiment 52, wherein the control sample comprising the first target further comprises said second target.

Embodiment 54. The method of any one of embodiments 46-53, further comprising obtaining a second target positive control fluorescent signal by repeating said method on a separate control sample containing said second target.

Embodiment 55. The method of Embodiment 54 wherein the control sample containing the second target contains the second target at a known concentration.

Embodiment 56. The method of Embodiment 54 or Embodiment 55, wherein said test sample further comprises said first target.

Embodiment 57. The method of any one of Embodiments 46-53, further comprising obtaining a fluorescent signal of a pooled positive control by repeating said method on a separate control sample containing said first and said second targets.

Embodiment 58. The method of Embodiment 57 wherein the pooled control sample contains a first target at a known concentration and/or a second target at a known concentration.

Embodiment 59. The method of any one of embodiments 50-58, further comprising normalizing said first fluorescent signal and/or said second fluorescent signal using any of said positive control fluorescent signal.

Embodiment 60. The method of any of Embodiments 46-59, further comprising obtaining a negative control fluorescent signal by repeating the method of any of Embodiments 1-3 on a separate negative control sample not containing:

(i) said first target; or

(ii) said second target; or

(iii) said first target or said second target.

Embodiment 61. The method of Embodiment 60 further comprising normalizing said first fluorescent signal and/or said second fluorescent signal using said negative control fluorescent signal.

Embodiment 62. The method of any of embodiments 46-61, further comprising comparing said first and/or second fluorescent signals to a threshold value, wherein:

- the threshold value is obtained using fluorescent signals obtained from the analysis of a series of samples tested in accordance with the method according to any of the embodiments 1-3, and including any one or more of:

(i) no template control and first target,

(ii) a no-matrix control and a second target,

(iii) no template control, first target and second target,

which allows you to determine the specified presence or absence of the first and second targets in the sample.

Embodiment 63. The method of Embodiment 62 wherein a series of samples are tested using said first closed stem-loop oligonucleotide at a known concentration and/or said second closed stem-loop oligonucleotide at a known concentration.

Embodiment 64. The method of any one of Embodiments 1-63, 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°С, 8°С, 9°С, 10°С, 11°С, 12°С, 13°С, 14°С, 15°С, 16°С, 17°С, 18°С, 19°С С, 20°С, 25°С, 30°С, 35°С, 40°С, 45°С, 50°С, 55°С, 60°С, 65°С, 70°С, 75°С, 80°C.

Embodiment 65. The method of any one of Embodiments 1-64, wherein any said amplicon is produced by any of polymerase chain reaction (PCR), strand displacement amplification (SDA), helicase dependent amplification (HDA), recombinase polymerase amplification (RPA), loop isothermal amplification (LAMP), rolling ring amplification (RCA), transcription-mediated amplification (TMA), self-sustaining sequence replication (3SR), nucleic acid sequence-based amplification (NASBA), or polymerase chain reaction with reverse transcription (RT-PCR).

Embodiment 66. The method of any one of Embodiments 1-65, wherein the sample is a biological sample obtained from a subject.

Embodiment 67. The method of any one of Embodiments 1-65, wherein the method is performed in vitro.

Embodiment 68. The method of any one of Embodiments 1-65, wherein the method is performed ex vivo.

Embodiment 69. A composition comprising:

first and second closed stem-loop oligonucleotides, wherein each of the closed stem-loop oligonucleotides contains a double-stranded stem fragment formed by hybridized nucleotides and connected to a single-stranded loop fragment formed by unhybridized nucleotides, wherein:

the number of hybridized nucleotides and/or

the sequence of hybridized nucleotides in the stem fragment of the first and second closed stem-loop oligonucleotides are different, and

each of the double-stranded stem fragments contains a fluorophore molecule attached to one strand and a quencher molecule attached to the opposite strand,

moreover, the fluorophore molecules emit in the same color region of the visible spectrum, and the melting temperature (Tm) of the double-stranded stem fragment of the first closed stem-loop oligonucleotide differs from the Tm of the double-stranded stem fragment of the second closed stem-loop oligonucleotide.

Embodiment 70. The composition of Embodiment 69 wherein the single stranded loop fragment of the first closed stem-loop oligonucleotide differs from the sequence of the single stranded loop fragment of the second closed stem-loop oligonucleotide.

Implementation option 71. Composition according to option 69 or 70, additionally containing:

a first MNKzyme containing substrate chains capable of hybridizing to a closed single-stranded loop fragment of a first closed stem-loop oligonucleotide; and

a second MNKzyme containing substrate chains capable of hybridizing to a single-stranded loop fragment of a second closed stem-loop oligonucleotide.

Embodiment 72. The composition of Embodiment 71, wherein:

substrate chains of the first MNCzyme hybridize with a single-stranded loop fragment of the first closed stem-loop oligonucleotide; and

the substrate chains of the second MNCzyme hybridize with a single-stranded loop fragment of the second closed stem-loop oligonucleotide.

Embodiment 73. The composition of Embodiment 72, wherein:

the first and/or second MNCzymes contain an aptamer sequence associated with the analyzed target compound, protein, compound or molecule, and sensor strands hybridized to the oligonucleotide sequence; and

the first MNKzyme is designed to detect a target that is different from the target of the second MNKzyme.

Embodiment 74. The composition of Embodiment 72, wherein:

the first and/or second MNKzymes contain sensory strands that hybridize to the target sequence, and the first MNKzyme is designed to detect a target that is different from the target of the second MNKzyme.

Embodiment 75. The composition of any one of Embodiments 69-74, further comprising:

a first oligonucleotide sequence hybridizing to said single-stranded loop fragment of a first closed stem-loop oligonucleotide that differs from the sequence of a single-stranded loop fragment of a second closed stem-loop oligonucleotide, thereby forming a double-stranded sequence comprising said first oligonucleotide;

a second oligonucleotide sequence hybridizing to said single-stranded loop fragment of a second closed stem-loop oligonucleotide different from the sequence of the single-stranded loop fragment of the first closed stem-loop oligonucleotide, thereby forming a double-stranded sequence comprising said second oligonucleotide;

a restriction endonuclease associated with a double-stranded sequence comprising said first oligonucleotide and capable of cleaving it; and/or

a restriction endonuclease associated with a double-stranded sequence comprising said second oligonucleotide and capable of cleaving it.

Embodiment 76. The composition of any one of Embodiments 69-75, further comprising:

a first double-stranded sequence comprising a first target oligonucleotide sequence hybridizing to said single-stranded loop fragment of the first closed stem-loop oligonucleotide different from the sequence of the single-stranded loop fragment of the second closed stem-loop oligonucleotide, thereby forming the first double-stranded sequence, containing the specified first oligonucleotide target;

a second double-stranded sequence comprising a second target oligonucleotide sequence hybridizing to said single-stranded loop fragment of the second closed stem-loop oligonucleotide different from the sequence of the single-stranded loop fragment of the first closed stem-loop oligonucleotide, thereby forming a double-stranded sequence containing said second target oligonucleotide;

the first primer oligonucleotide hybridizing to the first target oligonucleotide upstream (in the 5' direction) relative to said first double-stranded sequence, and the first enzyme having exonuclease activity (e.g. loop" at or near the end of the first oligonucleotide primer; and/or

a second primer oligonucleotide that hybridizes to the second target oligonucleotide upstream (in the 5' direction) relative to said second double stranded sequence, and a second enzyme having exonuclease activity (e.g. polymerase, exonuclease) associated with the loop fragment of the second closed stem-type oligonucleotide. loop" at the end of the second oligonucleotide primer

Embodiment 77. The composition of any one of Embodiments 69-76, further comprising:

a first double-stranded sequence comprising a first target oligonucleotide sequence hybridizing to said single-stranded loop fragment of the first closed stem-loop oligonucleotide different from the sequence of the single-stranded loop fragment of the second closed stem-loop oligonucleotide, thereby forming the first double-stranded sequence, containing the specified first oligonucleotide target;

a second double-stranded sequence containing a second target oligonucleotide sequence hybridizing to said single-stranded loop fragment of the second closed stem-loop oligonucleotide different from the sequence of the single-stranded loop fragment of the second closed stem-loop oligonucleotide, thereby forming a double-stranded sequence containing said second target oligonucleotide;

wherein the first enzyme having exonuclease activity is associated with the first double stranded sequence and the second enzyme having exonuclease activity is associated with the second double stranded sequence.

Embodiment 78. The composition of any one of Embodiments 69-77, further comprising:

the first DNAzyme and/or the first ribozyme hybridizing to a loop fragment of the first closed stem-loop oligonucleotide that differs from the sequence of the single-stranded loop fragment of the second closed stem-loop oligonucleotide,

a second DNAzyme and/or a second ribozyme hybridizing to a loop fragment of the second closed stem-loop oligonucleotide that differs from the sequence of the single-stranded loop fragment of the first closed stem-loop oligonucleotide,

cofactors for each said DNAzyme capable of conferring catalytic activity on each said DNAzyme and thus inducing the cleaving activity of any said DNAzyme hybridized to the loop fragment.

cofactors for each said ribozyme capable of conferring catalytic activity on each said ribozyme and thus inducing the cleavage activity of any said loop hybridized ribozyme.

Embodiment 79. The composition of any one of Embodiments 69-78, further comprising:

third closed stem-loop oligonucleotides, wherein said third closed stem-loop oligonucleotides comprise a double-stranded stem fragment of hybridized nucleotides connected to a single-stranded loop fragment of unhybridized nucleotides, wherein:

the number of hybridized nucleotides and/or

the sequence of hybridized nucleotides in the stem fragment of the third closed stem-loop oligonucleotides differs from the number and sequence of hybridized nucleotides in the stem fragments of the first and second closed stem-loop oligonucleotides, and

the double-stranded stem fragment of the third closed stem-loop oligonucleotides contains a fluorophore molecule connected to one strand and a quencher molecule connected to the opposite strand, and the fluorophore molecule of the third closed stem-loop oligonucleotide emits in a different region of the visible spectrum compared to with fluorophore molecules of the first and second closed stem-loop oligonucleotides.

Embodiment 80. The composition of any one of Embodiments 69-78, further comprising:

third closed stem-loop oligonucleotides, wherein said third closed stem-loop oligonucleotides comprise a double-stranded stem fragment of hybridized nucleotides connected to a single-stranded loop fragment of unhybridized nucleotides, wherein:

the number of hybridized nucleotides and/or

the sequence of hybridized nucleotides in the stem fragment of the third closed stem-loop oligonucleotides differs from the number and sequence of hybridized nucleotides in the stem fragments of the first and second closed stem-loop oligonucleotides, and

the double-stranded stem fragment of the third closed stem-loop oligonucleotides contains a fluorophore molecule connected to one strand and a quencher molecule connected to the opposite strand, and the fluorophore molecule of the third closed stem-loop oligonucleotide emits in the same region of the visible spectrum along compared with the fluorophore molecules of the first and second closed stem-loop oligonucleotides.

Embodiment 81. The composition of any one of Embodiments 69-78, further comprising:

third closed stem-loop oligonucleotides, wherein said third closed stem-loop oligonucleotides comprise a double-stranded stem fragment of hybridized nucleotides connected to a single-stranded loop fragment of unhybridized nucleotides, wherein:

the number of hybridized nucleotides and/or the sequence of hybridized nucleotides in the stem fragment of the third closed stem-loop oligonucleotides matches the number and sequence of hybridized nucleotides in the stem fragments of the first or second closed stem-loop oligonucleotides, and

the double-stranded stem fragment of the third closed stem-loop oligonucleotides contains a fluorophore molecule connected to one strand and a quencher molecule connected to the opposite strand, and the fluorophore molecule of the third closed stem-loop oligonucleotide emits in a different region of the visible spectrum compared to with fluorophore molecules of the first and second closed stem-loop oligonucleotides.

Brief description of the drawings

Preferred embodiments of the present invention will be described solely by way of example with reference to the appended Figures 1-8 below.

Figure 1: Typical LOCS reporters and melting temperatures (Tm) of their intact closed, blocked forms and split or collapsed open forms. Typical intact (closed) LOCS (FIG. 1A, LHS) have a loop region, a stem region, and a fluorophore (F)-quencher (Q) dye pair. Cleavage or destruction of the loop region can lead to the formation of open LOCS reporter structures (figure 1A RHS). The melting temperatures (Tm) of stem regions of intact LOCS exceed the Tm of stem regions in open LOCS structures. Essentially, stem A of LOCS A separates or melts at Tm 1. This is greater than Tm 2, which is the temperature at which stem A of the opened LOCS melts (FIG. 1B). Similarly, stem B of intact LOCS B melts at Tm 3. This is greater than Tm 4, which is the temperature at which stem B' of opened LOCS melts (FIG. 1B). The presence of a negative peak at a temperature corresponding to the Tm of the opened LOCS structure indicates the presence of a target that causes the specific LOCS to open. Because each stem in a given LOCS disclosed melts at a specific temperature, multiple LOCS labeled with the same fluorophore can be analyzed simultaneously in a single reaction.

Figure 2 shows a typical target detection strategy using LOCS oligonucleotides, which are versatile 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 contains a versatile substrate for a catalytic nucleic acid, such as MNKzyme, also known in the art as PlexZyme. MNCzymes are formed by aligning the sensory circuits of components - partzymes, which act as target sensors, next to each other on the target. The loop region of the LOCS oligonucleotide binds to the substrate-binding strands of the MNKzyme, and the MNKzyme cleaves the substrate within the loop, generating a fluorescent signal. The reaction mixture can be cooled, allowing the stem region of the opened LOCS to be reannealed, and then the reaction can be subjected to melt curve analysis, thereby monitoring fluorescence as the reaction mixture is heated. The presence of a melting peak corresponding to the Tm of the disclosed LOCS indicates the presence of a target that caused the assembly of MNKzyme. The target can be detected directly, or target amplicons obtained using target amplification protocols.

Figure 3 shows a typical target detection strategy using target-specific LOCS oligonucleotides. LOCS oligonucleotides contain a stem region, a fluorophore-quencher dye pair, and a loop region containing a region complementary to the target amplicon. In the scheme illustrated in Figure 3A, the loop region of LOCS oligonucleotides binds to target amplicons during amplification. During primer extension, polymerase exonuclease activity disrupts the loop region, generating a real-time fluorescent signal but leaving the stem regions intact. Stem regions are complementary to each other, but not to their target. After amplification, the reaction mixture can be cooled to re-anneal the stem of the broken open LOCS structure. Melting curve analysis can then be performed to measure the temperature at which the stem region derived from the expanded LOCS melts and generates fluorescence. In the scheme illustrated in Figure 3B, the loop region of the LOCS oligonucleotide contains a region complementary to the target amplicon and additionally contains a recognition site for a restriction enzyme, such as a nicking enzyme. The loop region of the LOCS oligonucleotide binds to the target, and the nicking enzyme cleaves the loop region, leaving the target molecule intact. This opens the LOCS and generates a fluorescent signal. The reaction mixture can then be cooled, allowing the stem of the split open LOCS structure to be re-annealed; and then a melt curve analysis can be performed to measure the temperature at which the stem region obtained from the disclosed LOCS melts. This strategy can be used to directly detect target sequences or detect target amplicons in combination with a target amplification method.

Figure 4 shows PCR amplification plots from reactions containing 10,000 copies (black line), 40 copies (gray line) or 0 copies (dashed line) of MgPa targets (Figure 4A), TV-Btub (Figure 4C) or both MgPa and TV-Btub (Figure 4E). Melting curve signatures obtained after amplification of reaction mixtures containing 10,000 copies (black line) and 40 copies (gray line) are shown for MgPa targets (Figure 4B), TV-Btub (Figure 4D), or both MgPa and TV-Btub (figure 4F). Melting signatures obtained by opening LOCS-1 in the presence of the MgPa target gene included peaks at melting points of 35°C and 41°C; melting signatures obtained by opening LOCS-2 in the presence of the TV-Btub target gene included a peak corresponding to a melting temperature of 50°C; while the melting signatures obtained by opening LOCS-1 and LOCS-2 in the presence of the MgPa and TV-Btub target genes included three peaks at melting temperatures of 35°C, 41°C and 50°C. The melting signature of LOCS in the presence of MgPa (Tm=35°C and 41°C) differs from the melting signature of LOCS obtained in the presence of the TV-Btub target gene (Tm=50°C). In addition, the LOCS melt signature in the presence of the MgPa and TV-Btub target genes (Tm=35° C., 41° C. and 50° C.) differs from the aforementioned melt signatures in the presence of only one target gene, indicating the detection of both targets.

Figure 5 (top panel) shows PCR amplification plots obtained for reaction mixtures containing 10,000 copies (black line), 40 copies (gray line) or 0 copies (dashed line) of the target genes MgPa (figure 5A1), HMPV ( figure 5 B1) and CT-ompA (figure 5C1). The results shown in the bottom panel are signatures of the melting curve obtained for the reaction mixture containing 10,000 copies (black line) and 40 copies (grey line) of the target genes MgPa (figure 5A2), HMPV (figure 5B2) and CT- ompA (Figure 5C2). These three targets were specifically detected using 3 different MNCzymes, each containing identical substrate-binding chains and differing only in target-binding chains. Each MNCzyme revealed the same universal LOCS oligonucleotide-1 containing a universal substrate and a universal stem. Each target produced a melting curve peaking at 40° C., which corresponds to the Tm of the universal stem.

Figure 6 (top panel) shows PCR amplification plots obtained for reaction mixtures containing 10,000 copies (black line), 40 copies (gray line) or 0 copies (dashed line) of TV-Btub target genes (Figure 6A1), VZV (figure 6 B1) and rpoB (figure 6C1). The results shown in the bottom panel are the signatures of the melting curve obtained for the reaction mixture containing 10,000 copies (black line) and 40 copies (gray line) of TV-Btub targets (Figure 6A2), VZV (Figure 6B2) and rpoB (Figure 6C2). These three targets were specifically detected using three different MNCzymes, each containing identical substrate-binding chains and differing only in target-binding chains. Each MNKzyme revealed the same universal LOCS-2 containing a universal substrate and a universal stem. Each target made it possible to obtain a melting curve with a peak at 50°C, which corresponds to the Tm of the universal stem in LOCS-2.

Figure 7 shows melt curve signatures obtained using LOCS-1 (Figure 7A), LOCS-2 (Figure TB), LOCS-3 (Figure 7C) and LOCS-4 (Figure 7D) and used to monitor the amplification of CT- Cds (figure 7A and 7C) or TFRC (figure 7B and 7D). The melting curve signatures obtained in the absence of a target (dashed line) contained peaks at Tm 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 presence of the CT-Cds target (black line; Figures 7A and 7C), the disclosed structures of LOCS-1 and LOC-3 allowed melting points of 30°C and 36°C, respectively. In the presence of the TFRC target (black line; Figures 7B and 7D), the disclosed structures of LOCS-2 and LOC-4 allowed melting points of 50°C and 32°C, respectively.

Figure 8 shows melt curves obtained for MgPa target amplification on a BioRad® CFX96 thermal cycler (Figure 8A), ABI 7500 (Figure 8B), Light Cycler 480 (Panel C) or XXpress PCR (Figure 8D). Both targets were tracked on the FAM channel on all devices.

Figure 9 shows melt curves obtained for amplification of the TV-Btub target on a BioRad® CFX96 thermal cycler (Figure 9A), ABI 7500 (Figure 9B), Lightcycler 480 (Figure 9C) or XXpress PCR (Figure 9D). Both targets were tracked on the FAM channel on all devices.

Figure 10 shows signatures of melting curves obtained from duplex PCR in which MgPa targets (black curves) and TV-Btub targets (gray curves) were co-amplified and read on the same channel. Solid and dotted lines represent target concentrations of 10,000 copies and 40 copies, respectively. Figure 10A shows melt curves obtained using LOCS-1 and LOCS-2 readings on the FAM channel; Figure 10B shows results obtained using LOC-5 and LOCS-6 reads on the HEX channel; Figure 10C shows the results obtained using the LOCS-7 and LOCS-8 reads on the Texas Red channel, and Figure 10D shows the results using the LOCS-9 and LOCS-10 reads on the Cy5 channel.

Figure 11 shows the melting curve signatures obtained after PCR amplification of reaction mixtures containing 10,000 copies of the target genes MgPa (Figure 11A), TV-Btub (Figure 11B), CTcry (Figure 11C), both MgPa and TV- Btub (figure 11D), both MgPa and CTcry (figure 11E) or both TV-Btub and CTcry (figure 11F). The melting signatures obtained by opening LOCS-1 and/or LOCS-2 and/or LOCS-11 produced peaks indicative of the melting of these structures at Tm ranging from 30° C. to 50° C., while intact closed LOCS melted in the Tm range from 64°C to 74°C. In each scenario, the LOCS melt signatures are unique and different from the melt signatures of other LOCS.

12 shows melt curve signatures obtained from PCR amplification of reaction mixtures containing 10,000 copies of each of the MgPa, TV-Btub and CTcry target genes. The melting signatures obtained by opening LOCS-1, LOCS-2 and LOCS-11 in the presence of the target genes MgPa, TV-Btub and CTcry included three peaks at melting temperatures of 30°C, 41°C and 49°C. A reaction mixture containing all three target genes can be distinguished from mixtures containing only one target gene (Figures 11A-11C) and mixtures containing two of the three target genes (Figures 11D-11E). For example, a reaction mixture containing all three target genes can be distinguished from a reaction mixture containing only TV-Btub and CTcry (Figure 11F) by the disappearance of the Tm peak of the disclosed LOCS 1 (~64°C).

Figure 13 shows melt curve signatures obtained from PCR amplification of 4-plex reaction mixtures containing 10,000 copies of each of the MgPa, TV-Btub, NGopa or gpd target genes. These four targets were specifically detected using 4 different MNCzymes, in turn, these MNCzymes were cleaved and revealed 4 different LOCS reporters tracked on 2 fluorescent channels. Each LOCS reporter contains a different universal substrate, but the same universal stems (stem-1 and stem-2) used on both the FAM channel and the Texas Red channel. The presence of the MgPa or TV-Btub genes was detected by an increase in the signal on the Texas Red channel; and the presence of the NGopa or gpd genes was detected by an increase in the signal on the FAM channel. Specific detection of MgPa or TV-Btub on the Texas Red channel and NGopa or gpd on the FAM channel was performed based on the presence of a unique melt curve signature. The melting signatures obtained on the Texas Red channel by opening LOCS-7 in the presence of MgPa and LOCS-8 in the presence of TV-Btub contained peaks at melting temperatures of 43°C and 53°C, respectively. The melting signatures obtained on the FAM channel by opening LOCS-12 in the presence of NGopa and LOCS-13 in the presence of gpd contained peaks at melting temperatures of 26°C, 42°C and 53°C, respectively.

Figure 14 shows the signatures of the melting curves obtained from single plex PCR, in which each of the loop regions of the three LOCS reporters contains a different substrate for its MNKzyme, however, all three LOCS contain the same stem sequence. Melting signatures obtained in the absence of a target (gray lines) show melting peaks at 74°C (Figure 14A), 75°C (Figure 14B) and 75°C (Figure 14C), corresponding to the melting temperatures of closed intact LOCS-2, LOCS-14 and LOCS-15, respectively. In the presence of the TFRC target (black line; Figures 14A, 14B and 14C), the disclosed structures LOCS-2, LOCS-14 and LOC-15 allowed melting points of 49°C, 48°C and 49°C, respectively.

Figure 15 shows melt curve signatures obtained using LOCS-16 (Figure 15A) and LOCS-17 (Figure 15B) to detect and identify two different targets (AF-NE-TV1 and AF-NE-R5b) using nicking endonuclease (Nt. AlwI) as an alternative to MNKzyme digestion. The melting curve signatures obtained in the absence of a target (gray lines) contained peaks at Tm 65°C and 76°C corresponding to the melting temperatures of closed intact LOCS-16 and LOCS-17, respectively. The melting curve signatures obtained in the presence of the target (black line; AF-NE-TV1 and AF-NE-R5b, respectively) contained peaks at Tm 29°C and 48°C corresponding to the melting points of the cleaved open LOCS-16 and LOCS- 17, respectively. The data from this example demonstrates that LOCS reporters can be used with alternative target detection methods, such as methods using nicking endonuclease enzymes.

Figure 16 shows amplification curves (Figure 16A) and melting curve signatures (Figure 16B) obtained using a strategy similar to that which mediates TaqMan signal generation/hydrolysis probes to open LOCS probes, namely disrupting specific regions of the probes (loops ) during PCR due to the exonuclease activity of the polymerase. PCR plots were generated using reaction mixtures containing 10,000 copies (black line) or 0 copies (gray line) of the TV-btub target gene (FIG. 16A). The signature of the melting curve obtained in the absence of a target (gray line) was characterized by a melting peak at Tm 61°C, which corresponds to the melting temperature of closed intact LOCS-18 (figure 16B). The signature of the melting curve obtained in the presence of the target (black line) was characterized by a melting peak at Tm 40°C, corresponding to the melting points of the split open LOCS-18 (figure 16B). The data from this example demonstrates that LOCS reporters can be used with alternative target detection methods, such as TaqMan technology, which uses enzymes with exonuclease activity to cleave probes that hybridize to amplicons.

Figure 17 shows PCR amplification plots obtained using reaction mixtures containing 20,000 copies of X/CTcry target (solid black line), 20,000 copies of Y/NGopa target (dashed black line), or 20,000 copies of both targets (gray line) at 39 °C (figure 17A) and 72°C (figure 17B). Threshold values of TX and TY are indicated for the amplification plot obtained at 39°C and 72°C, respectively. Fluorescence endpoints are indicated, designated as EX1, EX2, EY1 and EY2.

Figure 18 shows PCR amplification plots obtained using reaction mixtures containing 0 copies of X/CTcry target (solid black line), 32 copies of X/CTcry target (gray line) or 20,000 copies of both XCTcry targets (dashed black line) at 39°C with 20,000 copies of Y/NGopa target (Figure 18A), at 72°C with 20,000 copies of Y/NGopa target (Figure 18B), at 72°C with 20,000 copies of Y/NGopa target after FAF normalization (Figure 18C) , at 39°C with 32 copies of the Y/NGopa target (figure 18D), at 72°C with 32 copies of the Y/NGopa target (figure 18E), at 72°C with 32 copies of the Y/NGopa target after FAF normalization (figure 18F), at 39°C with 0 copies of Y/NGopa target (figure 18G), at 72°C with 0 copies of Y/NGopa target (figure 18H), at 72°C with 0 copies of Y/NGopa target after FAF normalization (figure 18I).

Figure 19 shows a non-linear logistic regression for the relative shift of the Cq value as a function of In number of copies of target Y (NGopa) - ln number of copies of target X (CTcry).

Figure 20 shows the melt curve characteristics obtained on the HEX channel from 6-plex reactions in one well using 10,000 copies of the gpd, gpd3 or porA target gene. Six targets were specifically detected and differentiated using two fluorescent channels (three targets per channel) in one well. These targets were detected using six different MNCzymes, which in turn cleaved and uncovered six different LOCS reporters. Figure 20A: LOCS-21 in the presence of gpd (53°C, 68°C and 85°C), figure 20B: LOCS-22 in the presence of gpd3 (30°C, 43°C, 77°C and 85°C), figure 20C: LOCS-23 in the presence of porA (68°C and 77°C), figure 20D: LOCS-21 and LOCS-22 in the presence of gpd and gpd3 (30°C, 43°C, 53°C and 85°C ), figure 20E: LOCS-21 and LOCS-23 in the presence of gpd and porA (53°C and 68°C), figure 20F: LOCS-22 and LOCS-23 in the presence of gpd3 and porA (30°C, 43°C , 68°С and 77°С).

Figure 21 shows melt curve signatures obtained from the Texas Red channel from the same 6-plex one-well reactions shown in Figure 20 containing 10,000 copies of the TV-Btub, MgPa or LGV target genes. In these reactions, six targets were specifically detected and differentiated using two fluorescent channels (three targets per channel). These targets were detected using six different MNCzymes, which in turn cleaved and uncovered six different LOCS reporters, when monitored on the HEX and Texas Red channels. Figure 21A: Melting curve signature obtained with LOCS-24 in the presence of TV-Btub (30°C, 42°C, 66°C and 82°C), Figure 21B: LOCS-25 in the presence of MgPa (53°C, 66°C and 82°C), figure 21C: LOCS-26 in the presence of LGV (68°C), figure 21D: LOCS-24 and LOCS-25 in the presence of TV-Btub and MgPa (30°C, 42°C, 53°C and 82°C), figure 21E: LOCS-24 and LOCS-26 in the presence of TV-Btub and LGV (30°C, 42°C and 68°C), figure 21F: LOCS-25 and LOCS-26 in the presence of MgPa and LGV (30°C, 43°C, 53°C and 68°C).

Figure 22 shows melting curve signatures obtained after PCR amplification from the same 6-plex reactions as shown in Figures 20 and 21 using 10,000 copies of each of gpd, gpd3 and porA (Figure 22A) or 10,000 copies each of the TV-Btub, MgPa and LGV target genes (FIG. 22B). These targets were detected using six MNCzymes, which in turn cleaved and opened six LOCS reporters, when monitored on two fluorescent channels. Figure 22A: Melting signature obtained on the HEX channel using LOCS-21, LOCS-22 and LOCS-23 in the presence of gpd, gpd3 and porA target genes (30°C, 43°C, 53°C and 68°C) . Figure 22B: Melting signature obtained on the TEXAS RED channel using LOCS-24, LOCS-25 and LOCS-26 in the presence of TV-Btub, MgPa and LGV (30°C, 42°C, 53°C and 68°C) .

Figure 23 shows melt curve signatures obtained from PCR amplification from 10-plex reactions using 10,000 copies of the target genes NGopa, porA, gpd, gpd3, TV-Btub, MgPa, CTcry, LGV, polA, or TFRC. Ten target genes were specifically detected using ten different MNCzymes, which in turn cleaved and uncovered ten different LOCS reporters, when monitored on five fluorescent channels (FAM, HEX, Texas Red, Cy5 and Cy5.5). Target differentiation on the FAM channel is shown in Figure 23A: LOCS-15 in the presence of NGopa (53°C), Figure 2B: LOCS-27 in the presence of porA (30°C and 76°C) and Figure 23C: LOCS-15 and LOCS-27 in the presence of both NGopa and porA (30°C and 53°C). Target differentiation on the HEX channel is shown in Figure 23D: LOCS-21 in the presence of gpd (53°C and 70°C), Figure 23E: LOCS-22 in the presence of gpd3 (30°C, 43°C and 76°C) and in figure 23F: LOCS-21 and LOCS-22 in the presence of target genes gpd and gpd3 (30°C, 43°C and 53°C). Target differentiation on the Texas Red channel is shown in figure 23G: LOCS-24 in the presence of TV-Btub (31°C, 41°C, 59°C and 83°C), in figure 23H: LOCS-28 in the presence of MgPa (63° C) and figure 231: LOCS-24 and LOCS-28 in the presence of both TV-Btub and MgPa (31°C, 41°C and 63°C). Target differentiation on the Cy5 channel is shown in Figure 23J: LOCS-29 in the presence of CTcry (61°C and 79°C), Figure 23K: LOCS-10 in the presence of LGV (47°C and 80°C) and Figure 23L: LOCS-29 and LOCS-10 in the presence of both CTcry and LVG (47°C and 61°C). Differentiation of targets on the Cy5.5 channel is shown in Figure 23M: LOCS-30 and LOCS-31 in the presence of both polA and TFRC gene targets (39°C and 59°C) and in Figure 23N: LOCS-31 in the presence of the TFRC gene (39°C and 80°C).

Figure 24 shows a comparative analysis of the level of differential fluorescence before and after amplification on the JOE channel using reaction mixtures containing 10,000 copies of TFRC, rpoB, pss or all combinations of two or more target genes, measured at 40.5°C, 56.5 °C and 78.5°C and expressed as the average of the fluorescence level from three replicate reactions minus the average of three replicate reactions of control without template. The margin of error is the standard deviation for every three repetitions.

Figure 25 shows the comparative differential level of fluorescence after amplification in the JOE channel using reaction mixtures containing 10,000 copies of TFRC, rpoB, pss, or all combinations of two or more target genes, measured at 32°C, 40.5°C, 56, 5°C and 65.5°C. Values were expressed as the difference in 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 over three replicates. The margin of error is the standard deviation for every three repetitions.

Figure 26 shows a comparative analysis of the differential level of fluorescence before and after amplification on the JOE channel using reaction mixtures containing 10,000 copies of TFRC, rpoB, pss or all combinations of two or more target genes, measured at 39.5°C, 56.5 °C and 65.5°C. The values were expressed as (i) the difference between the level of fluorescence before and after amplification at 39.5°C; (ii) the difference between the difference between the fluorescence level before and after amplification at 39.5°C and the difference between the fluorescence level before and after amplification at 39.5°C and the fluorescence level after amplification at 56.5°C and (iii) the difference between the difference between the fluorescence level before and after amplification at 56.5°C and the difference between the fluorescence level before and after amplification at 65.5°C, averaged over three replicates. The margin of error is the standard deviation for every three repetitions.

Figure 27 shows the comparative height of the melting peak at 40°C, 51°C and 65°C obtained on the JOE channel using reaction mixtures containing 10,000 copies of TFRC, rpoB, pss or all combinations of two or more target genes, averaged over three repeated reactions. The margin of error is the standard deviation for every three repetitions.

Definitions

In the present application, the singular forms include the mention of the plural forms, unless otherwise clearly follows from the context. For example, the phrase "polynucleotide" also includes many polynucleotides.

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

In this document, the term "multiple" means more than one. In some specific aspects or implementations, the set may mean 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 or more, and any integer derived from them, and any range derived from them.

As used herein, the term "subject" includes any animal of significant economic, social, or research value, including cattle, horses, sheep, primates, birds, and rodents. Therefore, a "subject" may be a mammal, such as a human, or a non-human mammal. The term also covers microbial subjects, including but not limited to bacteria, viruses, fungi/yeasts, protozoa, and nematodes. "Subject" in accordance with the present invention also includes infectious agents, such as prions.

As used herein, the terms "polynucleotide" and "nucleic acid" may be used interchangeably and refer to a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases, or analogs, derivatives, variants, fragments, or combinations thereof, including DNA, methylated DNA, alkylated DNA, RNA, methylated RNA, miRNA, miRNA, shRNA, mRNA, tRNA, snoRNA, mvRNA, mmRNA, microRNA precursors and primary microRNAs, other non-coding RNAs, ribosomal RNA, their derivatives, their amplicons, or any combination thereof, but not limited to. As a non-limiting example, the nucleic acid source can be selected from the group consisting of synthetic compounds, mammals, humans, animals, plants, fungi, bacteria, viruses, archaea, or any combination thereof. The terms "polynucleotide" and "nucleic acid", "oligonucleotide" include reference to any particular sequence, as well as to a sequence complementary to it, unless otherwise indicated.

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

As used herein, the term "oligonucleotide" refers to a segment of DNA, or a DNA-containing nucleic acid molecule, or RNA, or an RNA-containing molecule, or combinations thereof. Examples of oligonucleotides include target nucleic acids; substrates, for example, substrates that MNKzyme can modify; primers, for example primers used to amplify targets in vitro by methods such as PCR; and components of MNKzymes. The term "oligonucleotide" includes reference to any particular sequence, as well as a sequence complementary to it, unless otherwise indicated. The oligonucleotides may contain at least one addition or substitution, including a group containing 4-acetylcytidine, 5-(carboxyhydroxylmethyl)uridine, 2'-O-methylcytidine, 5-carboxymethylaminomethylthiouridine, dihydrouridine, 2'-O-methylpseudouridine, beta-D- galactosylkeosine, 2'-O-methylguanosine, inosine, N6-isopentenyladenosine, 1-methyladenosine, 1-methylpseudouridine, 1-methylguanosine, 1-methylinosine, 2,2-dimethylguanosine, 2-methyladenosine, 2-methylguanosine, 2-methylguanosine, 3-methylcytidine, 5-methylcytidine, N6-methyladenosine, 7-methylguanosine, 5-methylaminomethyluridine, 5-methoxyaminomethyl-2-thiouridine, beta-D-mannosylmethyluridine, 5-methoxycarbonylmethyluridine, 5-methoxyuridine, 2-methylthio-N6-isopentenyladenosine , N-((9-beta-ribofuranosyl-2-methylthiopurin-6-yl)carbamoyl)threonine, N-((9-beta-ribofuranosylpurin-6-yl)N-methylcarbamoyl)threonine, uridine-5-hydroxyacetic methyl ester acids, uridine-5-hydroxyacetic acid (v), vibutoxosin, pseudouridine, keosine, 2-thiocytidine, 5-methyl-2-thiouridine, 2-thiouridine, 4- thiouridine, 5-methyluridine, N-((9-beta-D-ribofuranosylpurin-6-yl)carbamoyl)threonine, 2'-O-methyl-5-methyluridine, 2'-O-methyluridine, vibutosin, 3-(3 -amino-3-carboxypropyl)uridine, but not limited to beta-D-arabinosyluridine, beta-D-arabinosylthymidine.

As used herein, the terms "complementary", "complementarity", "match", and "match" refer to the ability of nucleotides (e.g., deoxyribonucleotides, ribonucleotides, or combinations thereof) to hybridize to each other via Watson-Crick base pairing or ambiguous base pairing. Bonds can be formed by Watson-Crick base pairing between adenine (A) bases and uracil bases (U), between adenine bases (A) and thymine bases (T), between cytosine bases (C) and guanine bases (G). An ambiguous base pair is a base pair that does not follow the Watson-Crick rule and is formed between two nucleotides in a double-stranded polynucleotide structure (eg, guanine-uracil, inosine-uracil, inosine-adenine, and inosine-cytosine). Nucleotides referred to as "complementary" or being "complementary" to each other are nucleotides that have the ability to hybridize with each other, either by appropriate Watson-Crick base pairing or by ambiguous base pairing.

As used herein, the terms "non-complementary", "non-complementary", "mismatch", and "mismatch" refer to nucleotides (e.g., deoxyribonucleotides, ribonucleotides, and combinations thereof) that do not have the ability to hybridize to each other by Watson-Crick base pairing. or due to ambiguous base pairing.

As used herein, the term "enzyme" refers to any molecule capable of catalyzing a chemical reaction (eg, amplification of a polynucleotide, cleavage of a polynucleotide, etc.). Non-limiting examples of enzymes suitable for use in the present invention include nucleozymes and protein enzymes. Non-limiting examples of suitable nucleozymes include RNAzymes, MNKzymes, and DNAzymes. Non-limiting examples of suitable protein enzymes include exonucleases and endonucleases. Enzymes typically provide catalytic activity to facilitate one or more of the methods described herein. As a non-limiting example, the exonuclease activity may be the catalytic activity inherent in, for example, a polymerase. As a non-limiting example, endonuclease activity can be a catalytic activity inherent in, for example, a restriction enzyme, including a nicking endonuclease, a riboendonuclease, or a double-stranded structure-specific nuclease (DSN).

As used herein, the term "amplicon" refers to a nucleic acid (e.g., DNA or RNA, or combinations thereof) that is the product of natural or artificial amplification or replication of nucleic acids, including, but not limited to, PCR, RT-PCR, SDA, HDA, RPA, LAMP, RCA, TMA, 3SR or NASBA.

As used herein, the term stem-loop oligonucleotide is to be understood to mean DNA or a molecule containing DNA or RNA or a molecule containing RNA or a combination thereof (i.e. a hybrid molecule or a DNA-RNA complex) , containing or consisting of a double-stranded stem component connected to a single-stranded loop component. The double-stranded stem component contains a forward strand hybridized by pairing complementary bases with a complementary reverse strand, 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 component. loops. The double-stranded stem component may further comprise one or more fluorophores on one strand (eg, forward strand) and one or more quenchers on the opposite strand (eg, reverse strand).

In this document, the terms "stem-loop oligonucleotide" and "LOCS", also referred to herein as "LOCS oligonucleotide", "LOCS structure", "LOCS reporter", "intact LOCS", "closed LOCS" and "LOCS probes", are used interchangeably and mean a DNA or DNA-containing molecule, or RNA, or an RNA-containing molecule, or a combination thereof (i.e., a hybrid molecule or a DNA-RNA complex) containing or consisting of a double-stranded stem component (also referred to herein as "stem", "stem region" and "stem fragment") attached to a single-stranded loop component (also referred to herein as "loop", "loop region" and "loop fragment"). The double-stranded stem component contains a forward strand hybridized by pairing complementary bases with a complementary reverse strand, 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 component loops.

The double-stranded stem component may further comprise one or more fluorophores on one strand (eg, forward strand) and one or more quenchers on the opposite strand (eg, reverse strand). For example, the fluorophore(s) may be attached to or near the 5' end of the forward strand, and the quencher(s) may be attached to or near the 3' end of the reverse strand, or vice versa. The single stranded loop component may contain a region capable of serving as a substrate for a catalytic nucleic acid, such as MNKzyme, DNAzyme, ribozyme, apta-MNKzyme, or aptazime. In addition or alternatively, the single-stranded loop component may contain a region complementary to the target nucleic acid (e.g., targets for detection, quantification, etc.) and/or amplicons derived from it, which may additionally serve as a substrate for the enzyme. -exonucleases. As a non-limiting example, exonuclease activity may be inherent in a polymerase enzyme. In addition or alternatively, the single-stranded loop component region may contain a region that may: (i) be complementary to the target to be detected, (ii) contain one strand of a double-stranded site recognized by the restriction enzyme; and (iii) serve as a substrate for a restriction enzyme.

As used herein, the terms "disclosed stem-loop oligonucleotide", "disclosed LOCS", "disclosed LOCS oligonucleotide", "disclosed LOCS structure", "disclosed LOCS reporters", "disclosed LOCS probes", "disclosed LOCS", "cleaved LOCS", and "disrupted LOCS" are used interchangeably and refer to a "stem-loop oligonucleotide" or "LOCS" in which the single-stranded loop component is cleaved and/or disrupted (e.g., by an enzyme described herein ) so that at least one bond between adjacent nucleotides within the loop is removed, resulting in an open structure in the loop region. In the disclosed LOCS, the forward and reverse strands of the double stranded stem fragment may retain the ability to hybridize with each other to form a stem.

As used herein, the term "universal stem" refers to a double stranded sequence that can be included in any LOCS structure. The same "universal stem" can be used in LOCS containing loops containing catalytic nucleic acid substrates or a sequence complementary to the target in question. A single universal stem can be used as a surrogate marker for any target capable of facilitating the opening of a particular LOCS. A range of versatile stems can be incorporated into a range of LOCS designed to analyze any set of targets.

As used herein, the term “universal LOCS” refers to a LOCS structure comprising a “universal stem” and a “universal loop” containing a universal catalytic nucleic acid substrate that can cleave any MNCzyme with additional substrate-binding strands, regardless of target sensor strand sequences. as part of MNKzim. One generic LOCS can be used as a surrogate marker for any target capable of facilitating discovery of a particular LOCS. A range of versatile LOCSs can be incorporated into any multiplex analysis designed to analyze any set of targets.

In this document, the terms "nucleozyme", "catalytic nucleic acid", "nucleic acid with catalytic activity" and "catalytic nucleozyme" are used interchangeably and mean a DNA or DNA-containing molecule or complex, or RNA, or an RNA-containing molecule or complex, or a combination thereof (ie, a DNA-RNA-fusion molecule or complex) capable of recognizing at least one substrate and catalyzing the modification (eg, cleavage) of at least one substrate. Nucleotide residues in catalytic nucleic acids may contain the bases A, C, G, T and U, as well as their derivatives and analogues. The above terms include single molecular nucleozymes, which may contain a single DNA or DNA-containing molecule (also known in the art as a "DNA enzyme", "deoxyribozyme" or "DNAzyme") or RNA, or an RNA-containing molecule (also known in the art). as a "ribozyme" in the art) or a combination thereof, which is a DNA-RNA fusion molecule capable of recognizing at least one substrate and catalyzing the modification (eg, cleavage) of at least one substrate. The above terms include nucleozymes containing a DNA or a DNA-containing complex, or an RNA or an RNA-containing complex, or a combination thereof, which is a DNA-RNA-fusion complex capable of recognizing at least one substrate and catalyzing modification (e.g., cleavage) at at least one substrate. The terms "nucleozyme", "catalytic nucleic acid", "nucleic acid with catalytic activity", and "catalytic nucleozyme" include MNKzymes.

As used herein, the terms "MNKzyme" and "multiple nucleozyme" have the same meaning and refer to two or more oligonucleotide sequences (e.g., partzymes) that only in the presence of a compound that facilitates assembly of the MNKzyme (e.g., a target) form an active nucleozyme capable of catalytically modify the substrate. "MNKzyme" is also known in the art as "PlexZyme". MNCzymes can catalyze a number of reactions including substrate cleavage and other enzymatic modifications of the substrate or substrates. MNKzymes with endonuclease or cleaving activity are also known as "cleaving MNKzymes". Each of the partzyme components (partzymes A and B) binds to an assembly-facilitating compound (eg, a target DNA or RNA sequence) via Watson-Crick base pairing. MNKzyme is formed only when the sensor strands of partzymes A and B hybridize next to each other at a junction that facilitates assembly. MNKzyme substrate strands interact with a substrate whose modification (eg, cleavage) is catalyzed by the MNKzyme catalytic core formed by the interaction of the catalytic domains of partzymes A and B. MNKzymes can cleave DNA/RNA chimeric reporter substrates. Cleavage of a substrate between a pair of fluorophore and quencher dyes by MNKzyme can lead to the appearance of a fluorescent signal. The terms "multicomponent nucleozyme" and "MNKzyme" include two-component structures consisting of two molecules, or three-component structures consisting of three nucleic acid molecules, or other multicomponent structures, for example, formed by four or more nucleic acid molecules.

The terms "MNKzyme" and "multiple nucleozyme" as used herein are to be understood to encompass all known MNKzymes and modified MNKzymes, including compounds described in one or more of PCT Patent Publication Nos. WO/2007/041774, WO/2008/040095 , WO2008/122084 and related publications of US patents No. 2007-0231810, 2010-0136536 and 2011-0143338 (the contents of each of these documents are fully incorporated herein by reference). Non-limiting examples of MNZymes and modified MNZymes encompassed by the terms "MNZyme" and "multiple nucleozyme" include MNZymes with cleavage catalytic activity (provided herein), disassembled or partially assembled MNZymes containing one or more assembly inhibitors, MNZymes containing one or more aptamers (“apta-MNCzymes”), MNCzymes containing one or more truncated sensor chains and optionally one or more stabilizing oligonucleotides, MNCzymes containing one or more activity inhibitors, multicomponent inactive pronucleozymes (MHKi), each of which is detailed described in one or more of WO/2007/041774, WO/2008/040095, US 2007-0231810, US 2010-0136536 and/or US 2011-0143338.

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 only in the presence of a compound that facilitates assembly of the MNKzyme. described herein may co-form "MNKzyme". In some preferred embodiments, one or more, preferably at least two, partzyme components may comprise three regions or domains: a "catalytic" domain forming part of a catalytic core that catalyzes the modification; a "sensor chain" domain capable of associating with and/or binding to an assembly-facilitating compound; and a "substrate chain" domain capable of associating with and/or binding to a substrate. The terms "sensing circuit", "target sensor circuit" or "target sensing circuit" or "target circuit" can be used interchangeably to describe a partzyme domain that binds to an assembly facilitating compound, such as a target. Partzymes may contain at least one additional component, including but not limited to an aptamer, referred to herein as "apta-partzyme". A partzyme may contain several components, including, but not limited to, a partzyme component with a shortened sensory chain and a stabilizing chain component that stabilizes the structure of the MNKzyme through interaction with either a compound that facilitates assembly or a substrate.

The terms "assembly-facilitating molecule", "assembly-facilitating compound", "MNKzyme assembly-facilitating molecule" and "MNKzyme assembly-facilitating compound" as used herein refer to entities that can facilitate the self-assembly of partzyme components to form catalytically active MNKzyme through interaction with MNKzyme sensor circuits. As used herein, assembly-facilitating compounds may facilitate the assembly of MNCzymes having degrading or other enzymatic activity. In preferred embodiments, self-assembly of MNCzyme requires a connection to facilitate assembly. The assembly-facilitating compound may be a single molecule or may be comprised of two or more "assembly-facilitators" that can pair or bind to sensor strands of one or more oligonucleotide "partzymes". The assembly-facilitating compound may contain one or more of the nucleotide components lacking sequence complementarity with the MNKzyme sensor strand/s. A connection that facilitates assembly may be a target. The target may be a nucleic acid selected from the group consisting of DNA, methylated DNA, alkylated DNA, RNA, methylated RNA, miRNA, miRNA, shRNA, tRNA, mRNA, snoRNA, mvRNA, mmRNA, miRNA precursors and primary microRNAs, other non-coding RNA, ribosomal RNA, derivatives thereof, amplicons, or any combination thereof. The nucleic acid can be amplified. Amplification may include one or more of the following: PCR, RT-PCR, SDA, HDA, RPA, LAMP, RCA, TMA, 3SR, or NASBA.

As used herein, the term "detectable effect" is an effect that can be detected or quantified as an indicator of the cleavage of the LOCS probe/s that has occurred. The magnitude of the effect may indicate the amount of the original connection, for example, compounds that facilitate assembly (eg, target). The detectable effect can be detected by various methods, including fluorescence spectroscopy, surface plasmon resonance, mass spectroscopy, NMR, electron spin resonance, polarization fluorescence spectroscopy, circular dichroism, immunoassay, chromatography, radiometry, photometry, scintigraphy, electronic methods, electrochemical methods, UV spectroscopy, spectroscopy in the visible part of the spectrum or infrared spectroscopy, enzymatic methods, or any combination thereof.

As used herein, the terms "polynucleotide substrate" and "substrate" include any single-stranded or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases, or analogs, derivatives, variants, fragments, or combinations thereof, that can be recognized, modified, or acted upon by an enzyme, including catalytic nucleozyme. 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 "observable effect" for monitoring the catalytic activity of an enzyme. A "polynucleotide substrate" or "substrate" may be cleaved or degraded by one or more enzymes, including catalytic nucleozymes, e.g. limited to them.

As used herein, a "reporter substrate" is a substrate especially adapted to facilitate the measurement of substrate degradation or the appearance of a cleaved product of a catalyzed reaction. Reporter substrates can be in solution in a free or bound (or "attached") state, for example, to a surface or to another molecule. The reporter substrate can be labeled in any of a variety of ways, including, for example, fluorophores (with or without one or more additional components such as quenchers), radioactive labels, biotin (eg, by biotinylation), or chemiluminescent labels.

As used herein, a "universal substrate" is a substrate, such as a reporter substrate, that is recognized and catalyzed by multiple MNKzymes, each of which can recognize a different compound to facilitate assembly. The use of such substrates facilitates the development of separate assays to detect, identify, or quantify a wide range of assembly-facilitating compounds using structurally related MNCzymes, each of which recognizes a versatile substrate. Each of these versatile substrates can be independently labeled with one or more labels. In preferred embodiments, independently detectable labels are used to label one or more versatile substrates, which provides a convenient system for the independent or simultaneous detection of multiple assembly-facilitating compounds using MNKzymes. In some embodiments, the substrates may be catalytically modified by DNAzymes catalytically active in the presence of a cofactor, such as a metal ion cofactor, such as lead or mercury.

As used herein, the term "probe" 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 containing nucleozyme substrates in loop regions subject to catalytic cleavage by the nucleozyme.

The term "product" refers to a new molecule or molecules that are formed as a result of the enzymatic modification of the substrate. As used herein, the term "cleavage product" refers to a new molecule resulting from the cleavage or endonuclease activity of an enzyme. In some embodiments, the products of enzymatic cleavage or degradation of an intact closed LOCS structure contain two oligonucleotide fragments capable of hybridizing to form an open LOCS structure.

As used herein, the terms "melting point" and "Tm" in the context of a polynucleotide should be understood to refer to the melting point (Tm) calculated using Wallace's rule, according to which Tm=2°C (A+T)+4°C (G+ C) (see Wallace et al., (1979) Nucleic Acids Res. 6, 3543) unless otherwise stated. The effect of sequence composition on melting point can be understood using the nearest neighbor method, which is given by the following formula: Tm (°C)=ΔH° / (ΔS°+R ln[oligo]) - 273.15. In addition to stem length and sequence composition, other factors are known to influence melting temperature, including ionic strength and oligonucleotide concentration. A higher concentration of the oligonucleotide and/or ion increases the likelihood of the formation of a double-stranded structure, which leads to an increase in the melting point. Conversely, a lower concentration of oligonucleotide and/or ion promotes stem dissociation, resulting in a lower melting temperature.

As used herein, the term "quencher" includes any molecule that, when in close proximity to a fluorophore, receives radiant energy generated by the fluorophore and either dissipates the energy as heat or emits light at a wavelength longer than that of the fluorophore. Non-limiting examples of quenchers include Dabcyl, TAMRA, graphene, FRET fluorophores, ZEN quenchers, ATTO quenchers, black hole quenchers (BHQ), and Black Berry quenchers (BBQ).

It should be understood that herein the term "base" in the context of a nucleic acid has the same meaning as the term "nucleotide".

As used herein, the term "kit" refers to any material delivery system. Such delivery systems include systems that allow the storage, transport, or delivery of reagents (e.g., labels, reference samples, backing material, etc. in appropriate containers) and/or ancillary materials (e.g., buffers, written instructions for performing the assay, etc.). .d) from one place to another. For example, kits may contain one or more containers, such as boxes, containing the appropriate reagents and/or auxiliary materials. The term "set" includes both fragmented and combined sets.

As used herein, the term "fragmented kit" refers to a delivery system that includes two or more separate containers, each containing a portion of all components of the kit. The containers can be delivered to the intended recipient together or separately. Any delivery system that includes two or more separate containers, each containing a portion of all the components of the kit, is included in the meaning of the term "fragmented kit".

As used herein, the term "combination kit" refers to a delivery system containing all reaction assay components in a single container (eg, one box containing each of the required components).

It should be understood that the use herein of the term "about" in relation to the specified numerical value includes the specified numerical value and numerical values within plus or minus ten percent of the specified value.

It should be understood that the use of the term "between" herein when referring to a range of numerical values encompasses the numerical values at each endpoint of the range. For example, a 10 to 20 residue polypeptide includes a 10 residue polypeptide and a 20 residue polypeptide.

In this document, any description of documents describing the prior art, or statements derived from or based on these documents, is not an admission that the documents or statements derived from them are part of the general knowledge in the relevant field.

For purposes of description, all documents referenced herein are incorporated herein by reference in their entirety, unless otherwise noted.

Abbreviations

The following abbreviations are used throughout this document and throughout the specification:

LOCS: loop connected to stems

MNKzyme: multicomponent nucleozyme or nucleozyme consisting of several parts;

Partzyme: partial enzyme containing an oligonucleotide;

PCR: polymerase chain reaction;

gDNA: genomic DNA

NTC: Matrix Free Control

qPCR: quantitative real-time PCR

Ct; threshold cycle

^R2 ; correlation coefficient

nM; nanomolar

mm; millimolar

µl; microliter

dNTP; deoxyribonucleotide triphosphate

NF-H ₂ O: nuclease-free water;

LNA: closed nucleic acid;

F: fluorophore;

Q: damper;

N=A, C, T, G or any equivalent thereof;

N' = any nucleotide complementary to N or capable of forming a cN base pair;

(N): any number of N;

(N'): any number of N';

W: A or T;

R: A, G or AA;

rN: any ribonucleotide base;

(rN): any number of rN;

rR: A or G;

rY: C or U;

M: A or C;

H: A, C or T;

D: G, A or T;

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

FAM or 6-FAM: 6-carboxyfluorescein.

BHQ1: absorber - "black hole" 1

BHQ2: absorber - "black hole" 2

RT-PCR: reverse transcription polymerase chain reaction

SDA: strand displacement amplification

HDA: Helicase dependent amplification

RPA: recombinase polymerase amplification

LAMP: loop isothermal amplification

RCA: rolling ring amplification

TAPA: transcription-mediated amplification

3SR: Self-Sustaining Sequence Replication

NASBA: Amplification Based on Nucleotide Sequence

IB: Iowa Black® FQ

IBR: Iowa Black® RQ

shRNA: short hairpin RNA

miRNA: small interfering RNA

mRNA: messenger RNA

tRNA: transfer RNA

snoRNA: small nucleolar RNA

mvRNA: small transient RNA

mmRNA: small modulating RNA

pre-miRNA: precursor of microRNA

perv. microRNA: primary microRNA

LHS: left side

RHS: right side

DCO: double-stranded oligonucleotide

Tm: melting point

RFU: Relative Fluorescence Units.

Detailed description of the invention

The following detailed description provides information on exemplary embodiments of the present invention in sufficient detail to enable those skilled in the art to practice the present invention. The features or limitations of the various embodiments described do not necessarily limit other embodiments of the present invention or the present invention as a whole. Therefore, the following detailed description does not limit the scope of the present invention, which is defined solely by the claims.

The present invention relates to methods and compositions for improved multiplex detection of targets (eg, nucleic acids, proteins, analytes, compounds, molecules, etc.). Each of the methods and compositions uses LOCS oligonucleotides, which can be used in combination with various other agents.

- LOCS oligonucleotides

Exemplary LOCS oligonucleotides of the present invention are shown in Figure 1. Exemplary intact (closed) LOCS oligonucleotides (Figure 1A, LHS) comprise a loop region, a stem region, and a fluorophore (P)/quencher (Q) dye pair. Figure 1 shows two exemplary LOCS oligonucleotides, designated intact LOCS A and intact LOCS B. These LOCS oligonucleotides contain different loop sequences and different stem sequences. When used in combination, two given LOCS oligonucleotides of the present invention will typically differ in sequence and/or stem length. By way of a purely non-limiting example, stem A of intact LOCS A can be designed to melt at a first temperature, denoted as Tm 1; while stem B of intact LOCS B can be designed to melt at a different temperature, designated Tm 3 .

because intramolecular bonds are stronger than intermolecular bonds, stem regions of intact LOCS structures typically melt at higher temperatures than stem regions of open, cleaved, or disrupted LOCS oligonucleotide structures. For example, stem A of intact LOCS A melts at a Tm 1 greater than Tm 2, which is the temperature at which stem A' of open LOCS melts (FIG. 1B). Similarly, the melting temperature of stem B of intact LOCS B, which melts at Tm 3, is greater than Tm 4, which is the temperature at which stem B' of open LOCS melts (FIG. 1B). The presence of a peak (detected, for example, by an associated fluorescent marker) at a temperature corresponding to a particular disclosed LOCS structure indicates the presence of a target or target amplicons stimulating the opening of that particular LOCS structure. Because the expanded LOCS A stem melts at a different temperature than the opened LOCS B (FIG. 1B), multiple opened LOCS labeled with the same fluorophore can be simultaneously detected and analyzed in the same reaction mixture. In addition, multiple disclosed LOCS structures labeled with different fluorophores can also be simultaneously detected and analyzed in the same reaction mixture.

With reference to the exemplary embodiment depicted in Figure 2, the loop region sequence of the LOCS oligonucleotide may, for example, be a substrate of MNKzyme or other catalytic nucleic acid/acids. In additional embodiments shown in Figure 3A and Figure 3B, the loop region of the LOCS oligonucleotide may be a target-specific sequence fully or partially complementary to the target to be detected, which in the double-stranded state may be a substrate for degradation by an exonuclease, for example, exonuclease activity inherent in the polymerase. In yet another exemplary embodiment, shown in Figure 3B, the target-specific sequence within the loop may further comprise one strand of a double-stranded site recognized by the restriction enzyme. Hybridization of the loop sequence with the target sequence can result in a functional, cleavable restriction site. In preferred embodiments, the restriction enzyme is a nicking enzyme capable of cleaving the loop strand of the LOCS oligonucleotide leaving the target intact.

In some embodiments, the LOCS oligonucleotides of the present invention can be used for direct target detection. In other exemplary embodiments, LOCS can be used to detect target amplicons generated by target amplification technologies, including, but not limited to, PCR, RT-PCR, SDA, HDA, RPA, LAMP, RCA, TMA, 3SR, or NASBA. Cleavage or disruption leading to LOCS opening can occur in real time during target amplification or post-amplification, at the end point of the reaction. The loop region can be opened by target-dependent cleavage or degradation mediated by the enzymatic activity of a catalytic nucleic acid, including MNKzyme, DNAzyme, ribozyme; or the enzymatic activity of a protein enzyme, including but not limited to an exonuclease or an endonuclease. As a non-limiting example, the exonuclease activity may be the catalytic activity inherent in, for example, a polymerase. As a non-limiting example, endonuclease activity can be a catalytic activity inherent in, for example, a restriction enzyme, including nicking endonuclease, riboendonuclease, or duplex-specific nuclease (DSN).

An exemplary strategy in which the loop region contains a catalytic nucleic acid substrate is shown in Figure 2. In this strategy, LOCS oligonucleotides contain versatile substrates that can be used to detect any target. The LOCS oligonucleotide contains a stem region, a fluorophore/quencher dye pair, and an intermediate loop region containing a universal catalytic nucleic acid substrate, eg MNKzyme. MNKzyme can detect the target directly, or it can be used to detect amplicons generated during target amplification. MNCzymes are formed when sensory chains of partzymes acting as target sensors are aligned next to each other on the target or on target amplicons to form an active catalytic nucleus. The loop region of the LOCS oligonucleotide binds to the substrate-binding strands of the MNKzyme, and the MNKzyme cleaves the substrate within the loop, opening the LOCS and generating a fluorescent signal. The reaction mixture can then be cooled, allowing the stem of the opened LOCS structure to be reannealed. The reaction mixture can then be heated and a melt curve analysis performed to measure the temperature at which the stem region of the expanded LOCS melts. One of skill in the art would understand that targets can be detected in real time or after the completion of the reaction.

Reaction mixtures designed for the simultaneous detection of several targets may contain several LOCS oligonucleotides, each of which contains its own universal substrate inside the loops and its stem region, capable of melting at its own temperature after substrate/loop cleavage by various MNCzymes. The LOCS oligonucleotides may additionally contain the same fluorophore/quencher dye pairs. For example, MNKzyme 1 can be formed in the presence of target 1 and cleave substrate 1 within LOCS oligonucleotide 1. The result is a cleaved double-stranded open LOCS structure 1 containing stem 1 melting at temperature 1. Simultaneously, MNKzyme 2 can be formed in the presence of target 2 and cleave substrate 2 within LOCS oligonucleotide 2, resulting in a cleaved double-stranded open LOCS structure 2 containing stem 2, melting at temperature 2. When analyzing the melting profile of the reaction mixture, 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. Thus, one wavelength read on one channel of the device makes it possible to detect and distinguish between two targets.

The reaction mixture may further contain additional LOCS labeled with other fluorophore and quencher pairs. For example, LOCS oligonucleotides 1 and 2 can be labeled with fluorophore A, and LOCS oligonucleotides 3 and 4 can be labeled with fluorophore B. double-stranded open LOCS structure 1 containing stem 1 melting at temperature 1. MNKzyme 2 can form in the presence of target 2 and cleave substrate 2 within LOCS oligonucleotide 2, resulting in a cleaved double-strand opened LOCS structure 2 containing stem 2, melting at 2. MNKzyme 3 can be formed in the presence of target 3 and cleave substrate 3 within LOCS oligonucleotide 3, resulting in a cleaved double-stranded open LOCS structure 3 containing stem 3, melting at 3. MNKzyme 4 can be formed in the presence of target 4 and cleave substrate 4 inside the LOCS oligonucleotide 4, resulting in split double-stranded open LOCS structure 4 containing stem 4 melting at temperature 4. When analyzing the melting profile of the reaction mixture 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 analyzing the melting profile of the reaction mixture 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, such an analysis using two wavelengths read on two channels of the instrument allows four targets to be detected and distinguished. The skilled artisan will appreciate that this strategy can be extended to monitor cleavage of more than two targets at one particular wavelength, and furthermore, the number of fluorophores analyzed can be increased to a number determined by the maximum ability of the instrument to distinguish between individual wavelengths.

With reference to the exemplary embodiment depicted in Figure 3, two exemplary target detection strategies are illustrated using LOCS oligonucleotides containing and complementary target and/or amplicon specific loop regions. With respect to the embodiment shown in Figure 3A, the LOCS oligonucleotide comprises a stem region, a fluorophore-quencher dye pair, and a loop region containing a region complementary to the target amplicon. During amplification, the loop region of the LOCS oligonucleotides binds to the target amplicons. During primer extension, exonuclease activity of the polymerase disrupts the loop region, leaving stem regions intact and generating a real-time fluorescent signal. After amplification, the reaction mixture can be cooled, allowing the stem of the split open LOCS structure to be reannealed; and then a melting curve analysis can be performed to measure the temperature at which the stem region obtained from the collapsed opened LOCS melts. One of skill in the art would understand that targets can be detected in real time or after the completion of a reaction.

In a further exemplary embodiment depicted in Figure 3B, the LOCS oligonucleotide comprises a stem region, a fluorophore/quencher dye pair, and a loop region containing a region complementary to the target and additionally containing a recognition site for a restriction enzyme, e.g., a nicking enzyme. Upon binding of the loop region of the LOCS oligonucleotide to the target, the nicking enzyme cleaves the LOCS loop strand, leaving the target intact and generating a fluorescent signal. The reaction mixture can be cooled, allowing the intact stem of the split open LOCS structure to be re-annealed; and then a melt curve analysis can be performed to measure the temperature at which the stem region obtained from the split open LOCS melts. One skilled in the art will understand that the target can be directly detected in the reaction mixture without prior amplification. In addition, one skilled in the art would understand that target amplicons generated by target amplification can be detected in real time or after the reaction has ended. In preferred embodiments, the restriction enzyme used in combination with target amplification may be active and thermostable at the reaction temperature or temperatures.

In a further exemplary embodiment, the LOCS oligonucleotide may comprise a stem region, a fluorophore/quencher dye pair, and a loop region, which may contain a DNAzyme or ribozyme substrate, e.g., a DNAzyme or ribozyme, which can only be catalytically active in the presence of a metal ion. It is known in the art that specific DNAzymes and ribozymes require the presence of a cofactor metal cation to provide catalytic activity. For example, some DNAzymes and ribozymes can only be catalytically active in the presence of, for example, lead or mercury. Such metals may be present, for example, in an environmental sample. If the LOCS oligonucleotide contains a loop containing a substrate for a DNAzyme or ribozyme, for example lead-dependent, then the presence of lead in the sample can lead to cleavage of the LOCS and generation of a fluorescent signal. The reaction mixture can be cooled, allowing the intact stem of the split open LOCS structure to be re-annealed; and then a melt curve analysis can be performed to measure the temperature at which the stem region obtained from the split open LOCS melts. One skilled in the art will appreciate that multiple DNAzymes and/or ribozymes, each dependent on a particular cofactor metal, can be combined in a single reaction mixture designed to detect multiple targets, such as lead and mercury. In addition, one skilled in the art would appreciate that targets can be detected in real time or after the completion of a reaction.

As used herein, reference to a nucleotide sequence "substantially complementary" to another nucleotide sequence may mean that the first sequence is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the complementary strand 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.

As used herein, a nucleotide sequence "complementary" to another nucleotide sequence may mean that the first sequence is 100% identical to the complementary strand 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.

As used herein, reference to a nucleotide sequence "substantially non-complementary" to another nucleotide sequence may mean that the first sequence is less than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35% or 40% identical to the complementary strand 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. As used herein, a nucleotide sequence "not complementary" to another nucleotide sequence may mean that the first sequence is 0% identical to the complementary strand 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.

Non-limiting examples of target nucleic acids (i.e., polynucleotides) that can be detected using LOCS oligonucleotides may include DNA, methylated DNA, alkylated DNA, complementary DNA (cDNA), RNA, methylated RNA, miRNA, siRNA, shRNA, mRNA, tRNA, snoRNA, mvRNA, mmRNA, microRNA precursors and primary microRNAs, other non-coding RNAs, ribosomal RNA, their derivatives, their amplicons, or any combination thereof (including mixed polymers of deoxyribonucleotide and ribonucleotide bases).

In some embodiments, the melting point ("Tm") of the intact LOCS oligonucleotide is greater than the Tm of the disclosed LOCS structure.

Analysis of fluorescent signals

Fluorescent signals generated by dissociation of the disclosed LOCS structures can be analyzed by any suitable method for detecting, differentiating and/or quantifying target molecules in accordance with the methods of the present invention.

While standard melt curve analyzes can be used, various other approaches (see "Examples") are described and illustrated herein that can be used for analysis in various formats.

As a non-limiting example, at various times during the course of a reaction suitable for detecting cleavage or disruption of loop regions of LOCS oligonucleotides, fluorescence signal measurements can be made at one temperature or at multiple temperatures. As non-limiting examples, these times may include (i) a time at the start of a reaction and/or (i) one or more times during a reaction; and/or (iii) the moment of completion or end point of the reaction.

In some embodiments, the measurement of the fluorescent signal can be performed at two or more temperatures in each cycle during the PCR amplification. The analysis can be performed by comparing the levels of fluorescence obtained at the first and/or second temperature and/or at a different temperature.

In other embodiments, the fluorescent signal measurement may be performed at two or more temperatures in each cycle during PCR, and amplification curves may be plotted for each set of measurements obtained at each temperature. Each amplification plot can be assigned fluorescence thresholds for each specific temperature, and Cq values can be measured as the cycle number during which the amplification plots cross the thresholds. In embodiments where the fluorescent signal is measured at two temperatures in each cycle during the PCR, Cq measured using the fluorescent signal at the lower temperature may provide a direct quantification of the initial concentration of the first target; a Cq measured using a fluorescent signal at a higher temperature can be analyzed as shown in Example 12, allowing the initial concentration of the second target to be quantified.

As a non-limiting example, post-amplification fluorescence measurements compared to a no-template control at two temperatures, including a first temperature equal to or greater than the melting point of the first disclosed LOCS and inferior to the melting temperature of the second disclosed LOCS; and a second temperature equal to or greater than the melting point of the second disclosed LOCS allows specific detection of the first and second cleaved disclosed LOCS. As shown in Example 15 (Assay Method A), at the first temperature, cleavage of the first LOCS generates a significant fluorescence signal compared to the template-free control reaction mixture, crossing a predetermined threshold. At this first temperature, intact closed first and second LOCS and/or split open second LOCS do not significantly contribute to fluorescence signal production due to the higher melting temperatures of closed first and second LOCS and open second LOCS and their signal does not cross a predetermined threshold. . At a second temperature above the first temperature, second LOCS cleavage generates a significant fluorescence signal compared to a no-template control that crosses a predetermined threshold, while intact closed first and second LOCS and/or open first LOCS do not significantly contribute to fluorescence signal production. compared to the no-matrix control, and their signal does not cross a predetermined threshold. The detection of each cleaved LOCS reporter indicates the presence of the corresponding target in the sample.

As a non-limiting example, measurements of post-amplification fluorescence versus initial control fluorescence at specific temperatures, including a first temperature equal to or greater than the melting point of the first closed LOCS and less than the melting temperature of the second closed LOCS; and a second temperature equal to or greater than the melting point of the second closed LOCS allows the first and second cleaved open LOCS to be specifically detected.

As shown in Example 15 (Assay Method B), initial control fluorescence can be obtained by measuring fluorescence at or below a temperature at which none of the opened and/or closed LOCS generate a significant fluorescence signal (temperature-0). The analysis can be performed by comparing the levels of fluorescence obtained at the first and/or second temperature and temperature -0, and comparing these relative levels of fluorescence with a predetermined threshold. As shown in Example 15, at a first temperature, cleavage of the first LOCS generates a significant fluorescence signal compared to a signal at -0 that crosses a predetermined threshold. At this first temperature, intact closed first and second LOCS and/or split open second LOCS do not significantly contribute to fluorescence signal production and the signals do not cross a predetermined threshold. This is due to the higher melting points of the closed first and second LOCS and the open second LOCS. At a second temperature above the first temperature, splitting of the second LOCS generates a significant fluorescence signal compared to the signal obtained at -0 and/or at the first temperature, and the signal crosses a predetermined threshold. At this second temperature, the intact closed first and/or second LOCS and the opened first LOCS do not significantly contribute to fluorescence signal production compared to the signal at -0 and/or the first temperature, and the signal does not cross a predetermined threshold. The detection of each cleaved LOCS reporter indicates the presence of the corresponding target in the sample.

As a non-limiting example, a reference initial fluorescence can also be obtained by measuring the fluorescence at the first and second temperatures at additional time points when the reaction is initiated, for example, prior to PCR. At this additional time point, all LOCS are intact (closed), do not generate a significant fluorescence signal that does not cross a predetermined threshold. The analysis can be performed by comparing the levels of fluorescence obtained at the first and second temperatures at the time of initiation of the reaction, and the levels of fluorescence obtained at the first and second temperatures at the time during and/or after the reaction (for example, during PCR or after PCR ). As shown in Example 15 (Assay Method C), at the first temperature and time point after PCR, cleavage of the first LOCS generates a significant fluorescence signal compared to the intact closed first LOCS signal measured at the first temperature and time point before PCR. This relative signal crosses a predetermined threshold. At this first temperature, intact closed first and second LOCS and/or split open second LOCS do not significantly contribute to fluorescence signal production compared to pre-PCR signal due to the higher melting temperatures of closed first and second LOCS and open second LOCS. . At a second temperature above the first temperature and at a time point after PCR, cleavage of the second LOCS generates a significant fluorescence signal compared to the signal obtained at the second temperature at the time point before PCR. At this second temperature and at the time point after the PCR, the intact closed first or second LOCS and/or the opened first LOCS do not significantly contribute to the production of the fluorescence signal compared to the time point before the PCR at the same second temperature, and the signal does not cross the predetermined threshold . Alternatively, the difference between the relative signal obtained before and after PCR at the second temperature and the relative signal obtained before and after PCR at the first temperature can be compared to a predetermined threshold to determine the presence of a split second LOCS; moreover, in the presence of the disclosed second LOCS, the difference value exceeds a predetermined threshold, and in its absence, the difference value does not reach a predetermined threshold, as shown in example 15 (analysis method C). The detection of each cleaved LOCS reporter indicates the presence of the corresponding target in the sample.

As a non-limiting example, another alternative method is to measure the height of the melting peak in the melting signature, which is equivalent to the value of dFluorescence/dTemperature (dF/dT) at Tm of each split LOCS. However, the applicability of this method is not limited to determining the value of dF/dT at Tm split LOCS, but also includes the temperature range that includes Tm. The value of dF/dT at the first or second temperature (A°C) is equivalent to a fluorescence level gradient between (A - N)°C and (A+N)°C. As shown in Example 15 (analysis method D), the dF/dT value at the first temperature, calculated from the fluorescence signals at (first temperature + N)°C and (first temperature - N)°C, exceeds a predetermined threshold only in the presence of a disclosed first LOCS. The dF/dT value at the second temperature above the first temperature, calculated from the fluorescence signals at (second temperature + N)°C and (second temperature - N)°C, exceeds a predetermined threshold only in the presence of a disclosed second LOCS. The detection of each cleaved LOCS reporter indicates the presence of the corresponding target gene in the sample. dF/dT at the first and second temperatures can be expressed as a ratio unique to each of the possible combinations of disclosed and LOCS. This information can be used to find a particular disclosed LOCS, and therefore an appropriate target, by determining if the ratio falls within a predetermined range of values.

In some embodiments, the fluorescence signals or their derivatives at first, second and/or further temperatures are compared to a predetermined threshold, wherein the split open first LOCS signal crosses a predetermined threshold at a first temperature, the split open LOCS signal crosses a predetermined threshold at a second temperature, and the intact closed first and second LOCS signals do not cross predetermined thresholds at the first and second temperatures. Therefore, the practical value of predetermined thresholds at first and second temperatures can be used to detect split open first and second LOCS. The detection of split open first and second LOCS indicates the presence of the first and second target gene in the sample. In other embodiments, the fluorescence signals or their derivatives at the first, second and/or further temperatures can be expressed as a ratio, and there is a unique value for this ratio for each of the possible combinations of open and/or closed LOCS. These unique ratio values can be used to detect split open first and second LOCS and are indicative of the presence of the first and second target gene in the sample.

In some embodiments, detection, differentiation, and/or quantification of first and second targets using first and second LOCS on the same fluorescence channel is described. However, one of skill in the art should understand that these methods are applicable to the detection, differentiation and/or quantification of more than two targets, as shown in Example 15.

Typical Applications for LOCS Oligonucleotides

- Detection of targets during or after target amplification

The LOCS oligonucleotides of the present invention can be used to detect the presence of amplified target nucleotide sequences. There are no particular restrictions on the amplification methods in which LOCS reporters can be used. Amplicons generated from various reactions can be detected using LOCS reporters provided that the presence of target amplicons can cleave or destroy the LOCS reporter a to form open LOCS structures. Non-limiting examples of methods useful for cleaving or destroying loop regions contained in LOCS structures include cleavage with MNKzymes, DNAzymes, ribozymes, restriction enzymes, endonucleases, or degradation with exonucleases, including but not limited to the exonuclease activity of the polymerase.

Typically, nucleic acid amplification methods use enzymes (eg, polymerases) to make copies of a target nucleic acid specifically bound by one or more oligonucleotide primers. Non-limiting examples of amplification methods in which LOCS oligonucleotides can be used 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 isothermal amplification (LAMP), rolling ring amplification (RCA), transcription-mediated amplification (TMA), self-sustaining sequence replication (3SR), nucleic acid sequence-based amplification ( NASBA).

The qualified reader should understand that the uses of the LOCS oligonucleotides described above are provided solely as non-limiting examples. The disclosed LOCS oligonucleotides can be used in any primer-based nucleic acid amplification method, and the present invention is not limited to the specific embodiments described.

Detection of amplicons generated by LOCS reporters

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

Amplicons are generated using methods that use LOCS reporters that can be cleaved or destroyed 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.

MNKzyme 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. MNKzyme may contain one or more partzymes. MNCzymes are multicomponent nucleozymes that assemble and are catalytically active only in the presence of an assembly-facilitating compound, which may be, for example, a detectable target, such as an amplicon derived from a polynucleotide sequence using primers. MNCzymes consist of a plurality of "partial enzymes" or partzymes that self-assemble in the presence of one or more assembly-facilitating compounds to form active MNCzymes that catalytically modify substrates. The substrate and the assembly-facilitating compounds (target) are separate nucleic acid molecules. Partzymes contain several domains, including (i) sensory strands that bind to a compound that facilitates assembly (eg, a target nucleic acid); (ii) substrate strands that bind the substrate; and (iii) sequence fragments of the catalytic core, which, after assembly, combine to form a complete catalytic core. MNCzymes can be designed to recognize a wide range of compounds that facilitate assembly, including, for example, different target nucleotide sequences. In response to the presence of a compound that facilitates assembly, MNCzymes modify their substrates. This substrate modification may be associated with signal generation, and thus MNCzymes may generate an enzyme-enhanced output signal. The assembly facilitating compound may be a target nucleic acid present in a biological or environmental sample (eg, an amplicon derived from a target polynucleotide using primers). In such cases, detection of substrate modification due to MNKzyme activity indicates the presence of a target. Several MNCzymes are known in the art that are capable of cleaving nucleic acid substrates. MNCzymes and their modified forms are known in the art and are described in PCT Patent Publication Nos. WO/2007/041774, WO/2008/040095, WO2008/122084 and corresponding US Patent Publications Nos. 2007-0231810, 2010-0136536 and 2011-0143338 the contents of each of these documents are incorporated herein by reference in their entirety).

Diagnostic Applications

LOCS oligonucleotides can be used for diagnostic and/or prognostic purposes in accordance with the methods described herein. Diagnostic and/or prognostic methods can be performed ex vivo or in vitro. At the same time, the methods of the present invention need not be used for diagnostic and/or prognostic purposes, and therefore uses that are not diagnostic or prognostic are also contemplated by the present invention.

In some embodiments, the methods described herein can be used to diagnose an infection in a subject. For example, these methods can be used to diagnose a bacterial, viral, fungal/yeast, protozoan and/or nematode infection in a subject. In one embodiment, the implementation of the virus may be an enterovirus. The subject may be a bovine, equine, sheep, primate, bird, or rodent species. For example, the subject may be a mammal, such as a human, dog, cat, horse, sheep, goat, or cow. The subject may be suffering from a disease caused by an infection. For example, the subject may have meningitis caused by an enterovirus infection. Accordingly, in some embodiments, the methods of the present invention may be used to diagnose meningitis.

The methods of the present invention can be performed on a sample. The sample can come from any source. For example, the sample may be obtained from the environment, from an industrial source, or by chemical synthesis.

As used herein, the term "sample" should be understood to include a sample that has been modified from its original state, such as by purification, dilution, or the addition of any other component or components.

The methods of the present invention, including but not limited to diagnostic and/or prognostic methods, can be performed on a biological sample. A biological sample may be taken from the subject. In addition, stored biological samples can be used. Non-limiting examples of suitable biological samples include whole blood or a component thereof (e.g., blood cells, plasma, serum), urine, feces, saliva, lymph, bile, sputum, tears, cerebrospinal fluid, bronchoalveolar lavage fluid, synovial fluid, semen, ascites tumor fluid, breast milk and pus.

Sets

The present invention provides kits containing one or more of the agents for carrying out the methods of the present invention. Typically, kits for carrying out the methods of the present invention contain all the necessary reagents for carrying out the method.

In some embodiments, kits may contain oligonucleotide components capable of forming an MNCzyme in the presence of a suitable assembly-facilitating compound (eg, the amplicon described herein). For example, the kit may contain at least a first and second oligonucleotide component containing the first and second partzyme, and a second container containing the substrate, and the self-assembly of the first and second partzymes and the substrate in the MNKzyme requires the association of an assembly-facilitating compound (e.g., an amplicon) present in the tested sample. Accordingly, in such an embodiment, the first and second partzymes and the LOCS oligonucleotide containing the substrate in the loop region can be introduced into the test sample to determine the presence of one or more target amplicons. Typically, kits contain at least one LOCS oligonucleotide as provided herein.

Typically, kits of the present invention also contain other reagents, wash reagents, enzymes, and/or other reagents necessary to perform the methods of the present invention, such as PCR or other nucleic acid amplification methods.

Sets may be fragmented sets or combined sets as defined herein.

Fragmented kits contain reagents placed in individual containers and may contain small glass containers, plastic containers, or strips of plastic or paper. Such containers can provide efficient transfer of reagents from one compartment to another, while avoiding cross-contamination of samples and reagents, as well as quantitative addition of agents or solutions contained in each container from one compartment to another.

Such kits may also include a container into which the test sample is added, a container containing reagents used in the analysis, containers containing washing reagents, and containers containing a detection reagent.

Combination kits contain all of the reaction assay components in a single container (eg, a single box containing each of the required components).

The kit according to the present invention may also contain instructions for use of the components of the kit to perform the respective methods. The kits and methods of the present invention may be used in conjunction with automated analysis equipment and systems, for example, including but not limited to real-time PCR machines.

When amplifying, detecting, identifying, or quantifying different targets, one kit according to the present invention may be used, or, alternatively, different kits may be required, for example, containing reagents specific to each target. The methods and kits of the present invention find use in any circumstance in which it is desirable to detect, identify or quantify any object.

Those skilled in the art will appreciate that numerous changes and/or modifications can be made to the present invention, as described in terms of specific embodiments, without departing from the spirit of the present invention as described broadly. Thus, the present embodiments are to be considered in all respects as illustrative and not restrictive.

Examples

The present invention will be described in more detail with reference to the following specific examples, which should not be considered as limiting the invention in any way.

Example 1: Using LOCS reporters to increase the number of targets analyzed

In the following example, LOCS reporters were used to increase the number of targets that could be detected on a single fluorescence channel. In this example, two LOCS reporters were labeled 5' with a fluorophore (FAM) and 3' with a quencher. The loop regions of the LOCS reporters contained a substrate nucleic acid, and the stem regions contained a series of complementary base pairs holding the LOCS reporter in a stem-loop configuration. In this configuration, the fluorophore and quencher were in close proximity, and in the absence of a target, the fluorescence was quenched.

Oligonucleotides

Oligonucleotides specific to this experiment included: 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. Oligonucleotides specific for amplification and detection of MgPa were LOCS-1, partzyme A1, partzyme B1, forward primer 1 and reverse primer 1. Oligonucleotides specific for amplification and detection of TV-Btub were LOCS-2, Partzyme A2, Partzyme B2, forward primer 2 and reverse primer 2.

Reaction conditions

Real-time amplification and target sequence detection were performed in a total reaction volume of 20 μl using a BioRad® CFX96 thermal cycler. The cycle parameters were: 95°C for 2 minutes, 10 cycles of reduction, including 95°C for 5 seconds and 61°C for 30 seconds (0.5°C decrease per cycle) and 40 cycles, including 95 °C for 5 seconds and 52°C for 40 seconds (data obtained at the 52°C step). Melting curve parameters ranged from 20° C. to 90° C. in 0.5° C. increments and a 5 second hold (data logging was performed during the hold step). All reactions were performed in duplicate, reaction mixtures contained 40 nM each forward primer, 200 nM each reverse primer, 200 nM each partzyme A, 200 nM each partzyme B, 200 nM each LOCS reporter, 2 mM MgCl ₂ (Bioline) and 1x SensiFAST Probe No-ROX mixture (Bioline). The reaction mixtures contained a G-Block template (10,000 or 40 copies) homologous to the MgPa and/or TV-Btub genes, or did not contain a target (H ₂ O without nucleases (NF H ₂ O)).

results

Using the in vitro target amplification method known as PCR, two MNCzymes (MNCzyme 1 and MNCzyme 2) were used to monitor the amplification of target nucleic acids in real time by cleavage of the respective LOCS reporters (LOCS-1 and LOCS-2, respectively). MNKzyme 1 was designed to detect sequences homologous to the MgPa (Mycoplasma genitalium) gene and to cleave and open LOCS-1; and MNKzyme 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 MNCzymes, primers and LOCS oligonucleotides. The presence of MgPa or TV-Btub or both MgPa and TV-Btub (representing a co-infected sample) was detected by an increase in signal on the FAM channel.

The results shown in Figure 4 show the corresponding PCR amplification plots obtained using reaction mixtures containing 10,000 copies (black line), 40 copies (gray line) or 0 copies (dashed line) of MgPa targets (Figure 4A), TV -Btub (Figure 4C) or as MgPa, iac and TV-Btub (Figure 4E). Melting curve signatures obtained after amplification of reaction mixtures containing 10,000 copies (black line) and 40 copies (gray line) are shown for MgPa targets (FIG. 4B), TV-Btub (FIG. 4D), or both MgPa and TV- Btub (figure 4F). Results were means of duplicate reactions plotted using Microsoft Excel (version 14). The data obtained in this example demonstrate that the LOCS melt signatures obtained in the presence of the MgPa target gene (Tm=35°C and 41°C) differed from the LOCS melt signatures obtained in the presence of the TV-Btub target gene (Tm =50°C). In addition, the melting signatures of LOCS in the presence of the MgPa and TV-Btub target genes also differed from the aforementioned melting signatures obtained in the presence of only one target gene. In the presence of both targets, the melting curve signature (Tm=35°C, 41°C and 50°C) indicated the detection of both targets.

The data from this experiment also demonstrate that the melting signatures of each disclosed LOCS were reproducible for reaction mixtures containing high (10,000 gene copies) and low concentrations of the target (40 gene copies). This example demonstrates that two targets co-amplified in the same well using the same fluorescent channel can be distinguished based on their unique LOCS melting signatures. The example provides a simple method that can be used to detect multiple targets in a single well using a single fluorescence channel.

The melting temperatures (Tm) of the intact closed LOCS reporters were designed to exceed the annealing temperatures of the reaction mixture (52° C.) and these LOCS were sufficiently quenched during the reaction in the absence of target amplification. The Tm stem regions were designed to be unique for each LOCS reporter and this was achieved by varying the length and nucleotide composition of the complementary regions forming the stem. In this example, the data are consistent with the scenario that splitting the loop regions using MNCzymes leads to the opening of the LOCS, allowing it to be split into two fragments. Due to the large Tm difference between closed intact LOCS reporters and opened LOCS reporters, the opened LOCS fragments are not stable enough to maintain double strand formation at the annealing and acquisition temperature, and therefore a fluorescence signal is generated.

Real-time monitoring of the two target genes generated fluorescence curves that crossed an arbitrary threshold, yielding a value that may be known as Ct (Cycle Threshold). The observed fluorescence curve during the amplification step indicated the presence of one or both of the target genes in the sample. However, it is not possible to identify a specific target using the amplification curves alone. In this situation, a melting curve analysis was performed to determine which LOCS reporter was cleaved and thereby identify the specific target gene(s) present(s) in the sample. After the amplification step, the samples were subjected to a temperature gradient, with different melting curve characteristics corresponding to different universal stems. Due to the difference in Tm between open and intact LOCS reporters, stems of cleaved LOCS reporters dissociate at lower temperatures compared to uncleaved closed LOCS reporters. In addition, due to different stem lengths and base pair compositions, different stems (stem-1 and stem-2) within LOCS-1 and LOCS-2 produced unique fluorescent melting curve signatures.

Example 2: Using the same LOCS reporters to detect different target genes

In this example, two LOCS reporters (LOCS-1 and LOCS-2) were used to distinguish between many different genetic targets in order to demonstrate their versatility and applicability to the analysis of any target under consideration. When using PCR amplification to detect specific target genes and cleavage of the respective LOCS reporters (LOCS-1 and LOCS-2), several MNCzymes (MNCzymes 1-6) were used. Each of MNCzymes 1, 3, and 4 was able to cleave LOCS-1 in the presence of their respective target genes. Each of MNCzymes 2, 5, and 6 was able to cleave LOCS-2 in the presence of their respective target genes. This example shows the combinations of double strand structures shown in Table 1 for which amplification and detection are performed simultaneously in the same tube.

Oligonucleotides

Oligonucleotides specific to this experiment included: 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) , partzym A5 (SEQ ID NO: 19), partzym B5 (SEQ ID NO: 20), partzym A6 (SEQ ID NO: 21), partzym 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.

Components and reaction conditions.

Real-time amplification and target sequence detection were performed in a total reaction volume of 20 μl using a BioRad® CFX96 thermal cycler. The cycle parameters were: 95°C for 2 minutes, 10 cycles of reduction, including 95°C for 5 seconds and 61°C for 30 seconds (0.5°C decrease per cycle) and 40 cycles, including 95 °C for 5 seconds and 52°C for 40 seconds (data obtained at the 52°C step). Melting curve parameters ranged from 20° C. to 90° C. in 0.5° C. increments and a 5 second hold (data logging was performed during the hold step). All reactions were performed as duplex reactions containing the oligonucleotides shown in Table 1. Each reaction mixture 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 MgCl ₂ (Bioline) and 1x SensiFAST Probe No-ROX mix (Bioline). The reaction mixtures contained a G-Block template (10,000 or 40 copies) or did not contain a target (NF H ₂ O). The results shown in Figures 5 and 6 were duplicate mean values plotted with Microsoft Excel (version 14).

results

The results shown in Figure 5 (top panel) show PCR amplification plots obtained for reaction mixtures containing 10,000 copies (black line), 40 copies (gray line) or 0 copies (dashed line) MgPa (Figure 5A1), HMPV (figure 5 B1) and CT-ompA (figure 5C1). The results shown in the bottom panel are melt curve signatures obtained for reaction mixtures containing 10,000 copies (black line) and 40 copies (grey line) of the MgPa target genes (Figure 5A2), HMPV (Figure 5B2) and CT- ompA (Figure 5C2). These three targets were specifically detected using MNCzymes 1, 3, and 5, each containing target-specific sensory chains, but the substrate-binding chains for all of them were identical. Each MNKzyme, once formed in the presence of its assembly-facilitating target compound, was able to bind, cleave and open the same universal LOCS-1 containing the first universal substrate and the first universal stem. Thus, the presence of each target made it possible to obtain a melting curve with a peak at 40° C. corresponding to the Tm of the first universal stem.

The results shown in Figure 6 (top panel) show PCR amplification plots obtained for reaction mixtures containing 10,000 copies (black line), 40 copies (gray line) or 0 copies (dashed line) TV-Btub targets (Figure 6A1 ), VZV (figure 6B1) and rpoB (figure 6C1). The results shown in the bottom panel are the signatures of the melting curve obtained for reaction mixtures containing 10,000 copies (black line) and 40 copies (gray line) of TV-Btub targets (Figure 6A2), VZV (Figure 6B2) and rpoB ( figure 6C2). These three targets were specifically detected using MNCzymes 2, 4, and 6, each of which contained target-specific sensory chains, but the substrate-binding chains for all of them were identical. Each MNKzyme, once formed in the presence of its assembly-facilitating target compound, was able to bind, cleave and open the same universal LOCS-2 containing a second universal substrate and a second universal stem. Thus, the presence of each target made it possible to obtain a melting curve with a peak at 50° C. corresponding to the Tm of the second universal stem.

The data obtained in this example demonstrates that using the same universal LOCS reporters to detect different target genes produced comparable melting signatures. The LOCS melting signatures were shown to be independent of the target sequence and therefore easy to incorporate into any assay. This example demonstrates that these LOCS reporters are versatile and can be used to detect any desired gene or target transcript.

Example 3: Use of different stems to create LOCS reporters with different melting points to increase the number of targets analyzed

In this example, LOCS reporters containing stems of various compositions were used to demonstrate the effect of stem length and base pair composition on the final melt signature. 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 identical loop regions containing substrate 1; however, the LOCS-3 stem region (stem 3) was one base pair longer than the LOCS-1 stem region (stem 1), resulting in a structure with a slightly higher predicted Tm in both intact (closed) and open condition. MNKzymes with target-specific sensory chains capable of directing assembly in the presence of a target compound that facilitates assembly (AF-CT-Cds) and substrate chains capable of binding and cleaving substrate 1 in the LOCS-1 or LOCS-3 loop were used to isothermal signal detection monitoring

Similarly, LOCS-2 and LOCS-4 contained identical loop regions containing substrate 2; however, the LOCS-2 stem region (stem 2) was two base pairs longer than the LOCS-4 stem region (stem 4), resulting in a structure with a higher predicted Tm in both intact (closed) and open states . MNKzymes with target-specific sensor chains capable of directing assembly in the presence of a target-facilitating assembly compound (AF-TFRC) and substrate chains capable of binding and cleaving substrate 2 in the LOCS-2 or LOCS-4 loop were used for PCR monitoring. -amplifications.

Components and reaction conditions

Oligonucleotides specific to this experiment included: LOCS-1 (SEQ ID NO: 1), LOCS-2 (SEQ ID NO: 2), LOCS-3 (SEQ ID NO: 27), LOCS-4 (SEQ ID NO: 27). 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.

Real-time target sequence detection was performed in a total reaction volume of 20 μl. Each reaction mixture contained 1x NH4 buffer (Bioline), 8 mM MgCl ₂ (Bioline), 200 nM of each partzyme, and 200 nM LOCS reporter. The reaction mixtures contained the assembly-facilitating target compound at final concentrations of 10 nM or did not contain the target (control DNA). Reactions were incubated on a BioRad® CFX96 thermal cycler at 52°C, data was recorded every 10 seconds, the total number of cycles was 150. Then the samples were subjected to a temperature gradient in the range of 20°C-90°C with a step of 0.5°C and holding time 5 seconds (registration at each holding step). The results shown in Figure 7 were duplicate mean values plotted with Microsoft Excel (version 14).

results

The results presented in Table 2 and Figure 7 show differences in melting curve signatures obtained for closed intact LOCS reporters (dashed line) present in untargeted reactions and signatures consistent with the disclosed LOCS reporters (black line) in reaction mixtures containing targets that control the assembly of MNCzymes capable of cleaving and opening specific LOCS reporters.

In addition, the data obtained in this example demonstrates the difference in the melting signatures obtained by changing the sequence and/or length of the stem regions. The data demonstrates that the disclosed LOCS-1 produced melt signatures with a lower melting point (30° C.) compared to the temperature for the disclosed LOCS-3 (36° C.). Similarly, the disclosed LOCS-4 allowed to obtain melt signatures with a lower melting point (32°C) compared to the temperature for the disclosed LOCS-2 (50°C). These results are consistent with the fact that LOCS-1 and LOCS-4 contained shorter stems compared to LOCS-3 and LOCS-2, respectively. Moreover, each LOCS produced a unique melting signature, different from the signatures of other LOCS reporters. This experiment demonstrates that by varying the length and composition of the stem regions, different melt signatures could be obtained. Thus, a set of LOCS reporters can be used to simultaneously detect multiple targets on the same channel. In addition, the data obtained in this experiment was obtained using isothermal target detection, indicating that LOCS reporters can be used with a number of different biosensor analysis methods.

Example 4: Compatibility of LOCS reporters across multiple platforms

In this example, two LOCS reporters were tested for compatibility with several commonly used PCR platforms. Two MNKzymes (MNKzyme 1 and MNKzyme 2) were used to monitor the amplification of target nucleic acids in real time by cleavage of the respective LOCS reporters (LOCS-1 and LOCS-2, respectively). In this example, amplification and detection of two different genes, namely MgPa gene (Mycoplasma genitalium) and TV-Btub (Trichomonas vaginalis), were simultaneously performed in the same tube, both genes being able to generate a signal on the FAM channel.

Components and reaction conditions

Oligonucleotides specific to this experiment included: 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). Oligonucleotides specific for MgPa amplification and detection were LOCS-1, partzyme A1, partzyme B1, forward primer 1 and reverse primer 1.

Oligonucleotides specific for TV-Btub amplification and detection were LOCS-2, Partzyme A2, Partzyme B2, Forward Primer 2, Reverse Primer 1 and Reverse Primer 2. The sequences are listed in the Sequence Listing.

The cycle parameters were: 95°C for 2 minutes, 10 cycles of reduction, including 95°C for 5 seconds and 61°C for 30 seconds (0.5°C decrease per cycle) and 40 cycles, including 95 °C for 5 seconds and 52°C for 40 seconds (data obtained at the 52°C step). Melt curve parameters on a BioRad® CFX96 thermal cycler ranged from 20° C. to 90° C. in 0.5° C. steps with a 5 second hold (data logging was performed during the hold step). The melt curve parameters on the Lightcycler 480 thermal cycler were set to continuous data recording with a growth rate of 0.02°C from 20°C to 90°C with a hold of 1 second at each temperature. The parameters of the melt curve on an ABI 7500 thermal cycler were set as 1% growth rate in the range from 20°C to 90°C with a holding time of 5 s (data recording was performed during the holding step). The parameters of the melting curve on the Xxpress PCR thermal cycler were in the range from 30°C to 90°C with a step of 0.5°C and an exposure of 0.5 s (data recording was performed at the exposure stage). All reactions were performed in duplicate, reaction mixtures contained 40 nM each forward primer, 200 nM each reverse primer, 200 nM each partzyme A, 200 nM each partzyme B, 200 nM each LOCS reporter, 2 mM MgCl ₂ (Bioline) and lx SensiFAST Probe No-ROX mixture (Bioline). Real-time amplification and target sequence detection were performed in a total reaction volume of 20 μl, except for reactions run on an XXpress PCR thermal cycler, which were performed in a volume of 15 μl. The reaction mixtures contained a G-Block template (10,000 or 40 copies) or did not contain a target (NF H ₂ O). Real-time amplification and target sequence detection were performed using a BioRad® CFX96 thermal cycler, a Lightcycler 480 thermal cycler, an ABI 7500 thermal cycler, and an XXpress PCR thermal cycler.

results

The results shown in Figure 8 and Figure 9 show the melting curves obtained after amplification of the MgPa and TV-Btub targets, respectively, when amplifying on a BioRad® CFX96 thermal cycler (panel A) or a Lightcycler 480 thermal cycler (panel B) or an ABI 7500 thermal cycler (panel C) or XXpress PCR thermal cycler (panel D). Both targets were tracked on the FAM channel on all devices. The results illustrated in Figure 8D and Figure 9D were obtained using a 3 minute melt curve protocol; this demonstrates that melting curves can be quickly distinguished using LOCS reporters. These data demonstrate the possibility of obtaining comparable melt curve signatures on different platforms using two different LOCS reporters. In addition, the data demonstrates the possibility of obtaining comparable melt curve signatures using different melt curve parameters.

Example 5: LOCS Reporter Compatibility on Multiple Fluorescent Channels

In this example, ten LOCS reporters were tested for compatibility on several commonly used fluorescent channels. Two MNKzymes (MNKzyme 1 and MNKzyme 2) were used to monitor the amplification of target nucleic acids in real time by cleavage of the corresponding LOCS reporter. In this example, the same LOCS reporter sequences were used, however the LOCS were labeled with different fluorophore and quencher pairs. In this example, amplification and detection of two different genes, namely the MgPa gene (Mycoplasma genitalium) and TV-Btub (Trichomonas vaginalis), were simultaneously performed in the same tube, both genes being able to generate a signal on the same channel. Several fluorescent channels were tested, including FAM, HEX, Texas Red and Cy5.

Components and reaction conditions

Oligonucleotides specific to this experiment included: 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 were 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 were partzyme A2, partzyme B2, forward primer 2, reverse primer 2 and LOCS-2, LOCS-6, LOCS-8 or LOCS-10. The sequences are listed in the sequence listing.

The cycle parameters were: 95°C for 2 minutes, 10 cycles of reduction, including 95°C for 5 seconds and 61°C for 30 seconds (0.5°C decrease per cycle) and 40 cycles, including 95 °C for 5 seconds and 52°C for 40 seconds (data obtained at the 52°C step). Melt curve parameters on a BioRad® CFX96 thermal cycler ranged from 20° C. to 90° C. in 0.5° C. steps with a 5 second hold (data logging was performed during the hold step). All reactions were performed in duplicate, reaction mixtures contained 40 nM each forward primer, 200 nM each reverse primer, 200 nM each partzyme A, 200 nM each partzyme B, 200 nM each LOCS reporter, 2 mM MgCl ₂ (Bioline) and 1x SensiFAST Probe No-ROX mixture (Bioline). Real-time amplification and target sequence detection were performed in a total reaction volume of 20 μl using a BioRad® CFX96 thermal cycler. The reaction mixtures contained a G-Block template (10,000 or 40 copies) or did not contain a target (NF H ₂ O).

results

The results shown in Figure 10 show the melt curves obtained after amplification of the MgPa and TV-Btub targets, respectively, when amplified on a BioRad® CFX96 thermal cycler. Both targets were tracked simultaneously on the same fluorescent channel. Results shown in black illustrate melt curves obtained in the presence of the MgPa gene and results shown in gray illustrate melt curve signatures obtained in the presence of the TV-Btub target. Solid lines represent target concentrations of 10,000 copies and dotted lines represent target concentrations of 40 copies. The results shown in Figure 10A illustrate melt curves obtained using LOCS-1 and LOCS-2 on the FAM channel. Melting curves shown in Figure 10B were obtained using LOCS-5 and LOCS-6 on the HEX channel. The melt curves shown in Figure 10C were obtained using LOCS-7 and LOCS-8 on a Texas Red channel, and the melt curves shown in Figure 10D were obtained using LOCS-9 and LOCS-10 on a Cy5 channel. The data demonstrated the possibility of obtaining comparable melt curve signatures on different fluorescence channels using the same two stems and LOCS substrates, by simply changing the fluorophore-quencher pair.

Example 6: Simultaneous detection of three targets on one fluorescent channel using three LOCS reporters

In the following example, LOCS reporters were used to increase the number of targets that could be detected on a single fluorescence channel. In this example, three LOCS reporters were labeled 5' with a fluorophore (FAM) and 3' with a quencher. The loop regions of the LOCS reporters contained a nucleic acid substrate, and the stem regions contained a series of complementary base pairs holding the LOCS reporter in a loop-stem configuration. In this configuration, the fluorophore and quencher were in close proximity, and in the absence of a target, the fluorescence was quenched.

Oligonucleotides

Oligonucleotides specific to this experiment included: 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. Oligonucleotides specific for amplification and detection of MgPa were LOCS-1, partzyme A1, partzyme B1, forward primer 1 and reverse primer 1. Oligonucleotides specific for amplification and detection of TV-Btub were LOCS-2, Partzyme A2, Partzyme B2, Forward Primer 2 and Reverse Primer 2. Oligonucleotides specific for CTcry amplification and detection were LOCS-11, Partzyme A9, Partzyme B9, Forward Primer 7 and Reverse Primer 7.

Reaction conditions

Real-time amplification and target sequence detection were performed in a total reaction volume of 20 μl using a BioRad® CFX96 thermal cycler. The cycle parameters were: 95°C for 2 minutes, 10 cycles of reduction, including 95°C for 5 seconds and 61°C for 30 seconds (0.5°C decrease per cycle) and 35 cycles, including 95 °C for 5 seconds and 52°C for 40 seconds (data obtained at the 52°C step). Melting curve parameters ranged from 20° C. to 90° C. in 0.5° C. increments with a 5 second hold (data logging was performed during the hold step). All reactions were performed in duplicate, reaction mixtures contained 40 nM each forward primer, 200 nM each reverse primer, 200 nM each partzyme A, 200 nM each partzyme B, 200 nM each LOCS reporter, 2 mM MgCl ₂ (Bioline) and 1x SensiFAST Probe No-ROX mixture (Bioline). The reaction mixtures contained a G-Block template (10,000 copies) homologous to the MgPa and/or TV-Btub and/or CTcry genes, or did not contain a target (NF H ₂ O).

results

Using the in vitro target amplification method known as PCR, three MNCzymes (MNCzyme 1, MNCzyme 2 and MNCzyme 9) were used to monitor the amplification of target nucleic acids in real time by cleaving the respective LOCS reporters (LOCS-1, LOCS-2 and LOCS-11, respectively). MNKzyme 1 was designed to detect sequences homologous to the MgPa (Mycoplasma genitalium) gene and to cleave and open LOCS-1; MNKzyme 2 was designed to detect sequences homologous to TV-Btub (Trichomonas vaginalis) and cleave and open LOCS-2; and MNKzyme 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 MNCzymes, primers and LOCS oligonucleotides. The presence of MgPa, TV-Btub, CTcry, or various combinations of these three targets (representing a multiple infection sample) was detected by an increase in signal on the FAM channel.

The results shown in figures 11 and 12 show the corresponding melting curve signatures obtained after amplification in reaction mixtures containing 10,000 copies of targets MgPa (figure 11A), TV-Btub (figure 11B), CTcry (figure 11C), both MgPa and and TV-Btub (Figure 11D), both MgPa and CTcry (Figure 11E), both TV-Btub and CTcry (Figure 11F), or all three targets of MgPa, TV-Btub and CTcry (Figure 12). Results were means of duplicate reactions plotted using Microsoft Excel (version 14).

The data obtained in this example and summarized in Table 3 demonstrate that the LOCS melt signatures obtained in the presence of the MgPa target gene (Tm=39°C and 66°C) differed from the LOCS melt signatures obtained in the presence of the target genes. TV-Btub (Tm=49°C and 64°C) and CTcry (Tm=30°C, 41°C and 64°C). In addition, the LOCS melt signatures in the presence of two or more target genes also differed from the aforementioned melt signatures obtained in the presence of only one target gene. In the presence of MgPa and TV-Btub targets in the same reaction mixture, the unique melting curve signature (Tm=39°C, 49°C and 66°C) indicated the detection of both targets. Similarly, in the presence of MgPa and CTcry or TV-Btub and CTcry in the same reaction mixture, unique melting curve signatures ((Tm=31°C, 41°C and 74°C) and (Tm=30°C, 41°C, 49°C and 66°C), respectively) indicated the detection of two targets and allowed them to be identified. In the presence of MgPa, TV-Btub and CTcry targets in one reaction mixture, the unique melting curve signature (Tm=30°C, 41°C and 49°C) indicated that all three targets were detected.

This example demonstrates that three targets co-amplified in the same well using the same fluorescent channel can be distinguished based on their unique LOCS melting signatures. 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 the different stem length and base pair composition, the different stems (stem-1, stem-2, and stem-3) within LOCS-1, LOCS-2, and LOCS-11 produced unique fluorescence melting curve signatures.

In this particular example, the Tm of open LOCS 1 (one peak at ~39°C indicates MgPa) and open LOCS 3 (two peaks at ~30/31°C and ~41°C indicate CTcry) results in a combined peak in the presence of both MgPa targets and CTcry targets. However, the reaction mixture containing MgPa, TV-Btub and CTcry (figure 12) can be distinguished from the reaction mixture containing only TV-Btub and CTcry (figure 11F) by the disappearance of the Tm peak of the disclosed LOCS 1 (~64°C ). Although the pooled peak is not ideal for this method, alternative stem sequences that produce more distinct peaks are easily identified using the stem screening assay described in Example 7.

Example 7: Method for Screening Sequences Suitable for Use as Generic Stems that Can be Inserted into LOCS Reporter Oligonucleotides

In the following example, the suitability of a series of double-stranded oligonucleotides (DSOs) for use as universal stems was tested. These non-looped DCOs were subjected to gradually increasing temperatures in the presence of SYBR green I intercalating dye. LOCS reporter oligonucleotides as universal stems. SYBR green I is an intercalating dye that generates a fluorescence signal when bound to double-stranded DNA structures. Under the influence of a gradiently increasing temperature, two LCO oligonucleotide chains dissociate, and fluorescence decreases. The following example demonstrates that the temperature at which two oligonucleotides (Tm) dissociate can be controlled by changing the stem length and composition. In addition, DCO was tested at various concentrations to demonstrate how Tm can be further manipulated by changing the concentration of oligonucleotides.

Oligonucleotides

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

Reaction conditions

Melt curve analysis was performed in a total reaction volume of 20 μl using a BioRad® CFX96 thermal cycler. Melting curve parameters ranged from 15° C. to 70° C. in 0.5° C. increments with a 5 second hold (data logging was performed during the hold step). All reactions were performed in duplicate, with reaction mixtures containing 100 nM, 200 nM or 300 nM LCO-A and LCO-B oligonucleotides, 1 μM SYBR green I, 8 mM MgCl ₂ (Bioline) and 1x NH4 buffer (Bioline).

results

The results shown in Table 4 show the melting points (Tm) obtained by subjecting the DCO to gradient increasing temperatures. Results were means of duplicate reactions plotted using Microsoft Excel (version 14).

This example demonstrates that increasing stem length and GC content increased Tm. Moreover, increasing the concentration of the oligonucleotide also slightly increased Tm, with the maximum difference observed when the concentration was increased from 100 nM to 200 nM. The method described in this example can be used as a screening method to improve the identification of alternative stem sequences, allowing you to get more clearly distinguishable peaks.

This method can also be used to study the effect 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, it is possible to identify a number of DTCs that, when included in LOCS, provide a "ladder" of clearly separated Tm. In an alternative format, the DCO can be labeled with various fluorophore and quencher pairs to investigate the ability of different dye combinations to affect the Tm of a particular DCO.

Example 8: Simultaneous detection of four targets in one well using two fluorescent channels and four LOCS reporters using only two stems

In the following example, several LOCS reporters containing stems of the same composition attached to different substrates (loops) were combined in a single reaction vessel to demonstrate the ability to reuse the same stem region to simultaneously detect and discriminate multiple targets in one reaction mixture. In the current example, two stems (stem-1 and stem-2) were used to discriminate four different targets on two fluorescent channels. In this example, two LOCS reporters were labeled 5' with the FAM fluorophore (LOCS-12 and LOCS-13) and the other two were labeled 5' with the Texas Red fluorophore (LOCS-7 and LOCS-8). All four LOCS reporters were labeled 3' with quencher. Each of the loop regions of the four LOCS reporters contained its own nucleic acid substrate (Sub1, Sub2, Sub3, and Sub4). Two LOCS reporters, one for each fluorophore, contained stem-1 (LOCS-7 and LOCS-12) and the other two LOCS reporters contained stem-2 (LOCS-8 and LOCS-13). The stem regions contained a series of complementary base pairs holding the LOCS reporter in a stem-loop configuration. In this configuration, the fluorophore and quencher were in close proximity, and in the absence of a target, the fluorescence was quenched. Stem-1 had a lower melting point than stem-2 and was attached to loops 1 and 3 to detect MgPa and NGopa targets at the first detection temperature (40°C), while stem-2 had a higher melting point and was attached to loops 2 and 4 to distinguish between TV-Btub and gpd targets at a second detection temperature (50°C).

Oligonucleotides

Oligonucleotides specific to this experiment included: 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 BIO (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) and reverse primer 9 (SEQ ID NO: 55). The sequences are listed in the sequence listing. Oligonucleotides specific for amplification and detection of MgPa were LOCS-7, partzyme A1, partzyme B1, forward primer 1 and reverse primer 1. Oligonucleotides specific for amplification and detection of TV-Btub were LOCS-8, Partzyme A2, Partzyme B2, Forward Primer 2 and Reverse Primer 2. Oligonucleotides specific for NGopa amplification and detection were LOCS-12, Partzyme A10, Partzyme B10, Forward Primer 8 and Reverse Primer 8. Oligonucleotides specific for to amplification and detection of gpd were LOCS-13, partzyme A11, partzyme B11, forward primer 9 and reverse primer 9.

Reaction conditions

Real-time amplification and target sequence detection were performed in a total reaction volume of 20 μl using a BioRad® CFX96 thermal cycler. The cycle parameters were: 95°C for 2 minutes, 10 cycles of reduction, including 95°C for 5 seconds and 61°C for 30 seconds (0.5°C decrease per cycle) and 35 cycles, including 95 °C for 5 seconds and 52°C for 40 seconds (data obtained at the 52°C step). Melting curve parameters ranged from 20° C. to 90° C. in 0.5° C. increments and a 5 second hold (data logging was performed during the hold step). All reactions were performed in duplicate, reaction mixtures contained 40 nM each forward primer, 200 nM each reverse primer, 200 nM each partzyme A, 200 nM each partzyme B, 200 nM each LOCS reporter, 2 mM MgCl ₂ (Bioline) and 1 × SensiFAST Probe No-ROX mix (Bioline). The reaction mixtures contained a G-Block template (10,000 copies) homologous to the MgPa, TV-Btub, NGopa, or gpd genes, or did not contain a target (NF H ₂ O).

results

Using the in vitro target amplification method known as PCR, four MNCzymes (MNCzyme 1, MNCzyme 2, MNCzyme 10, and MNCzyme 11) were used to monitor amplification of target nucleic acids in real time by cleavage of the respective LOCS reporters (LOCS-7, LOCS -8, LOCS-12 and LOCS-13, respectively). MNKzyme 1 was designed to detect sequences homologous to the MgPa (Mycoplasma genitalium) gene and to cleave and open LOCS-7; MNKzyme 2 was designed to detect sequences homologous to TV-Btub (Trichomonas vaginalis) and cleave and open LOCS-8; MNKzyme 10 was designed to detect sequences homologous to NGopa (Neisseria gonorrhoeae) and cleave and open LOCS-12; and MNKzyme 11 was designed to detect sequences homologous to gpd (herpes simplex virus type 2) and cleave and open LOCS-13. In this experiment, amplification and detection were performed in one tube containing all MNCzymes, primers and LOCS oligonucleotides.

The presence of the MgPa or TV-Btub genes was detected by an increase in the signal on the Texas Red channel; and the presence of the NGopa or gpd genes was detected by an increase in the signal on the FAM channel. The distinction between MgPa or TV-Btub on the Texas Red channel and NGopa or gpd on the FAM channel was performed based on the presence of a unique melt curve signature. The results shown in Figure 13A show the corresponding melting curve signatures obtained on the Texas Red channel after amplification in reaction mixtures containing 10,000 copies of the target gene MgPa (black line) or TV-Btub (gray line).

The results shown in Figure 13B show the corresponding melting curve signatures obtained from the FAM channel after amplification in reaction mixtures containing 10,000 copies of the target gene NGopa (black line) or gpd (gray line). Results were means of duplicate reactions plotted using Microsoft Excel (version 14). The presence of MgPa was determined both by the increase in the Texas Red signal and by the unique melting signature on the Texas Red channel (Tm=43°C). The presence of TV-Btub was determined both by the increase in the Texas Red signal and by the unique melting signature on the Texas Red channel (Tm=54°C). The presence of NGopa was determined both by the increase in the FAM signal and by the unique melting signature on the FAM channel (Tm=26°C and 42°C). The presence of gpd was determined both by the increase in the FAM signal and by the unique melting signature on the FAM channel (Tm=49°C).

These data, obtained in this example and summarized in table 5, demonstrate the possibility of reusing the same universal stem regions in one reaction due to their attachment to different substrates (loops) and detection on different fluorescent channels. In addition, the data demonstrates that four targets co-amplified in one well when monitored on two fluorescent channels can be distinguished based on their unique LOCS melting points. The example shows a simple method that can be used to detect multiple targets in a single well. Using the same stem sequence to discriminate multiple targets in a single reaction mix facilitates easy design of highly multiplex assays and allows quick and easy tailoring of LOCS probes to detect any chosen target gene.

Example 9 The same stem sequence can be combined with different substrates in LOCS reporters to obtain reproducible melting points.

In the following example, three LOCS reporters were used to demonstrate the ability to pair the same stem with different loop sequences (substrates) to obtain comparable melt signatures. In this example, all three LOCS reporters were labeled 5' with FAM fluorophore and 3' with quencher. Each of the loop regions of the three LOCS reporters contained a different nucleozyme substrate (MNKzyme), but all three LOCS contained the same stem sequence (stem-2).

Oligonucleotides

Oligonucleotides specific to this experiment included: 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: 2). 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. Oligonucleotides specific for amplification of the TFRC gene were forward primer 10 (SEQ ID NO: 72) and reverse primer 10 (SEQ ID NO: 73). Oligonucleotides specific for TFRC amplicon detection and LOCS-2 digestion were partzyme A8 (SEQ ID NO: 31) and partzyme B8 (SEQ ID NO: 32). Oligonucleotides specific for TFRC amplicon detection and LOCS-14 digestion were partzyme A12 (SEQ ID NO: 74) and partzyme B12 (SEQ ID NO: 75). Oligonucleotides specific for TFRC amplicon detection and LOCS-15 digestion were partzyme A13 (SEQ ID NO: 76) and partzyme B13 (SEQ ID NO: 77).

Reaction conditions

Real-time amplification and target sequence detection were performed in a total reaction volume of 20 μl using a BioRad® CFX96 thermal cycler. The cycle parameters were: 95°C for 2 minutes, 10 cycles of reduction, including 95°C for 5 seconds and 61°C for 30 seconds (0.5°C decrease per cycle) and 35 cycles, including 95 °C for 5 seconds and 52°C for 40 seconds (data obtained at the 52°C step). Melting curve parameters ranged from 20° C. to 90° C. in 0.5° C. increments and a 5 second hold (data logging was performed during the hold step). All reactions were performed in duplicate, reaction mixtures contained 40 nM each forward primer, 200 nM each reverse primer, 200 nM each partzyme A, 200 nM each partzyme B, 200 nM each LOCS reporter, 2 mM MgCl ₂ (Bioline) and 1x SensiFAST Probe No-ROX mixture (Bioline). The reaction mixtures contained a G-Block template (10,000 copies) homologous to the human transferrin receptor gene (TFRC) or did not contain a target (NF H ₂ O).

results

In this experiment, PCR amplification, signal detection, and melting curve differentiation were performed in the same tube as a one-plex reaction. During PCR, three MNCzymes (MNCzyme 8, MNCzyme 12 and MNCzyme 13) were used to monitor the amplification of target nucleic acids in real time by cleaving the respective LOCS reporters (LOCS-2, LOCS-14 and LOCS-15, respectively). All three MNCzymes were designed to detect the same target sequence (TFRC gene), however, each MNCzyme could hybridize to a different substrate sequence (loop) and cleave it, thereby revealing a different LOCS reporter.

The results shown in Figure 14 show the corresponding melting curve signatures obtained on the FAM channel after PCR amplification in reaction mixtures containing 10,000 copies of the TFRC target gene (black line) or not containing the target (gray line). The presence of the TFRC gene was demonstrated by the presence of a PCR amplification curve crossing a threshold (data not shown) and melting peaks at Tm 49°C, 48°C and 49°C (Figures 14A, 14B and 14C, respectively) correlated with cleaved LOCS -2, LOCS-14 and LOCS-15, respectively. The absence of the TFRC gene was determined by the absence of a PCR amplification curve crossing the threshold (data not shown) and the presence of a melting peak at Tm 74°C, 75°C and 75°C, correlated with uncleaved LOCS-2, LOCS-14 and LOCS - 15, respectively (figures 14A, 14B and 14C, respectively). Results were means of duplicate reactions plotted using Microsoft Excel (version 14). The data obtained in this example demonstrates the possibility of using the same universal stem regions with different substrates (loops) to obtain comparable melt curve signatures. Using the same stem sequence to construct LOCS reporters with different substrates facilitates easy design of highly multiplex assays and allows LOCS probes to be quickly and easily adapted to detect any chosen target gene.

Example 10 - Use of Nicking Endonuclease as an Alternative Method for Deploying LOCS Reporters

In the following example, a nicking endonuclease (Nt. Alwl) was used to demonstrate the production of comparable melting signatures in the opening of a loop fragment of LOCS reporters after nicking endonuclease digestion as an alternative to MNKzyme digestion. The general strategy is illustrated in Figure 3B. In this example, the double labeled LOCS reporter contained a loop region complementary to the target sequence and a stem region not complementary to the target. Hybridization of the loop region of the LOCS reporter with the target completed the formation of the nicking endonuclease recognition site, facilitating cleavage of the LOCS reporter by the nicking endonuclease and leaving the target intact. Since intramolecular bonds are stronger than intermolecular bonds, the stem regions of intact LOCS structures melt at higher temperatures than the stem regions of open split LOCS structures.

Oligonucleotides

Oligonucleotides specific to this experiment included: 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.

Reaction conditions

Real-time target sequence detection was performed in a total reaction volume of 20 μl using a BioRad® CFX96 thermal cycler. The reaction mixtures were incubated at a constant temperature of 52°C, data was collected every 20 seconds, a total of 200 times. Melting curve parameters ranged from 20° C. to 90° C. in 0.5° C. increments with a 5 second hold (data logging was performed during the hold step). All reactions were performed in duplicate, the reaction mixtures contained 5 mm MgCl ₂ (Ambion), 1x buffer II for PCR (ABI), 4 units of Nt. Alwl (NEB) and 200 nM LOCS (LOCS-16 or LOCS-17). Reaction mixtures contained AF-NE-TV1 or AF-NE-R5b (200 nM) or no target (NF H20).

results

Signal detection and melting curve differentiation were performed in one tube. The results shown in Figures 15A and 15B show the respective signatures of the LOCS-16 and LOCS-17 melting curves obtained on the FAM channel in reaction mixtures containing 200 copies of the target (black line, AF-NE-TV1 or AF-NE-R5b, respectively), or not containing a target (grey line, NF H2O). Results were means of duplicate reactions plotted using Microsoft Excel (version 14). The presence of the AF-NE-TV1 target was detected both by a rapid increase in fluorescence over time and by the presence of a melting peak at 29°C corresponding to cleaved LOCS-16. The absence of AF-NE-TV1 target was determined by (i) no increase in fluorescence over time, (ii) no melting peak at 29°C, and (iii) presence of a melting peak at 65°C corresponding to uncleaved LOCS-16 (Figure 15A) .

The presence of the AF-NE-R5b target was detected both by a rapid increase in fluorescence over time and by the presence of a melting peak at 48°C corresponding to cleaved LOCS-17. The absence of AF-NE-R5b target was determined by (i) no increase in fluorescence over time, (ii) no melting peak at 48°C, and (iii) presence of a melting peak at 76°C corresponding to uncleaved LOCS-17 (Figure 15B) .

The data from this example demonstrates that LOCS reporters can be used with alternative methods of target detection, such as methods using nicking endonuclease enzymes. In addition, the data also demonstrates that LOCS reporters containing the same stem (stem-2) produce peaks at the same melting temperature (~50°C) regardless of the mechanism of loop disruption, such as nickel cleavage. enzyme or MNKzyme. In this example, intact and opened LOCS reporters containing stem-2 allowed to obtain melting peaks at 48°C and 76°C, respectively. These melting peaks are comparable to the melting peaks obtained using stem-2 with MNCzymes, as shown in Figure 4D. Although small shifts of 1-2°C may be observed for LOCS containing the same stem when used in different protocols, e.g., loop digestion with nicking enzymes compared to loop digestion with MNCzyme, these shifts likely reflect differences in the reaction medium. mixtures, where the Tm of the stem can be affected by the concentration of salt, glycerol, or another component.

Example 11 - Use of TaqMan exonuclease as an alternative method for opening LOCS reporters

In the following example, a method similar to the TaqMan probe/hydrolysis probe was used to demonstrate the formation of comparable melting signatures upon opening of the stem fragment of LOCS reporters after exonuclease digestion, as an alternative method to MNKzyme digestion of the loop. The general strategy for this example is illustrated in Figure 3A. In this example, the double labeled LOCS reporter contained a loop region complementary to the target sequence and a stem region not complementary to the target. During PCR amplification, the loop region of the LOCS reporter could hybridize to the target sequence and/or amplicon. During the primer extension step of PCR, the 5' primer is extended into the hybridization region of the LOCS reporter with the target, the polymerase gradually cleaves the 5' end of the loop region of the LOCS reporter, but leaves the stem region intact. Breaking the loop region lowers the melting point of the LOCS stem because the intermolecular forces between two LOCS stem fragments are much weaker than the intramolecular forces that arise between them within a closed LOCS molecule.

Oligonucleotides

Oligonucleotides specific to this experiment included: 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.

Reaction conditions

Real-time amplification and target sequence detection were performed in a total reaction volume of 20 μl using a BioRad® CFX96 thermal cycler. The cycle parameters were: 95°C for 2 minutes, 50 cycles at 95°C for 5 seconds and 52°C for 40 seconds (data recorded at the 52°C step). Melting curve parameters ranged from 20° C. to 90° C. in 0.5° C. increments with a 5 second hold (data logging was performed during the hold step). All reactions were performed in duplicate, reaction mixtures contained 400 nM of each forward primer, 200 nM LOCS-16, 2 mM MgCl ₂ (Bioline) and 1x SensiFAST Probe No-ROX mix (Bioline). The reaction mixtures contained the G-Block synthetic template (10,000 copies) or did not contain the target (NF H ₂ O).

results

In this experiment, PCR amplification, signal detection and melting curve differentiation were performed in the same tube. The presence of the target gene (TV-btub) was determined by the presence of a PCR amplification curve and a melting peak at Tm 40° corresponding to disrupted open LOCS-18. The absence of the target gene was determined by the absence of a PCR amplification curve, the absence of a melting peak at Tm 40°C, and the presence of a melting peak at Tm 61°C corresponding to uncleaved closed LOCS-18. The results shown in Figures 16A and 16B show the corresponding amplification curves and melting curve signatures obtained on the FAM channel in reaction mixtures containing 10,000 copies of the target gene (black line) or not containing the target (gray line). Results were means of duplicate reactions plotted using Microsoft Excel (version 14).

The data from this example demonstrates that LOCS reporters can be used with alternative target detection methods using mechanisms similar to the TaqMan probe/hydrolysis probe, namely mechanisms in which enzymes with exonuclease activity degrade sequences that hybridize to amplicons. Essentially, in this example, the LOCS probe consisted of a loop fragment that performed the same function as the standard TaqMan probe sequence, however, the "TaqMan-like probe sequence" contained complementary stem sequences attached to the 5' and 3' hybridization region. , which are not in themselves complementary to the target, but are complementary to each other. In addition, the data also demonstrate that LOCS reporters containing the same stem (stem-1) produce peaks at the same melting point regardless of the mechanism of loop disruption, eg, exonuclease disruption or MNKzyme cleavage. In this example, intact and opened LOCS reporters containing stem-1 allowed to obtain melting peaks at 61°C and 40°C, respectively. These melting peaks are comparable to the melting peaks obtained using stem-1 with MNCzymes, as shown in Figures 5A2, 5B2 and 5C2. The current method has the advantage over other exonuclease based methods using a melt curve, such as TOCE, that the released labeled probe fragment does not have to subsequently hybridize to the capture probe to detect the presence of the target sequence, and the LOCS reporter can better be quenched at the initial stage, since the fluorophore and quencher are fixed in close proximity to each other.

Example 12 Method for Simultaneous Detection and Quantification of Multiple Targets on a Single Fluorescent Channel Using Two Acquisition Temperatures During Amplification

The following example demonstrates an approach where LOCS reporters allow simultaneous detection and quantification of multiple targets on a single fluorescence channel by recording fluorescence readings at two discrete temperatures in real time during PCR. This strategy eliminates the need for PCR melt curve analysis as demonstrated in the various examples above. In addition, this example describes two alternative data analysis methods that facilitate the quantification of any target if it is present in the sample.

In this example, two LOCS reporters containing stems of different lengths, compositions, and melting points were used to simultaneously detect, distinguish, and quantify two X and Y targets on the same fluorescence channel by measuring real-time fluorescence at two different temperatures (T _L and T _H ) during each PCR cycle. The assay was designed such that Target X was tracked by LOCS-X (cleaved only by MNAzyme X in the presence of Target X) and Target Y was tracked by LOCS-Y (digested by MNAzyme Y only in the presence of Target Y). In addition, the assay was designed such that the disclosed LOCS-X had a lower Tm compared to the disclosed LOCS-Y.

The lower detection temperature (T _L ) was chosen so that the open LOCS-X stem would melt (dissociate), resulting in increased fluorescence, while the closed LOCS-X stem and both opened and closed LOCS-Y stems remained in the associated and extinguished state. The higher temperature (T _H ) was chosen such that the stem of the opened LOCS-Y melted (dissociated), resulting in increased fluorescence, while the stem of the closed LOCS-Y remained in an associated and quenched state. Because the LOCS-X stem had a lower Tm, it melted (dissociated) at this higher temperature resulting in increased fluorescence whether it was in its open or closed conformation; however, the fluorescence of the closed LOCS-X only contributed to the increase in the background/original signal.

Using fluorescence measurements performed at temperatures T _L and T _H , two PCR amplification curves can be constructed. Thresholds for determining the presence of X and/or Y targets were set for both the T _L plot (threshold X; TX) and the T _H plot (threshold Y; TY). Thresholds (TX and TY) and various endpoints at which reactions are known to plateau can be predetermined based on previous experiments where reactions containing only target X plateaued at the EX1 endpoint at T _L or at the end point EX2 at T _H ; reactions containing the Y target plateaued at the EY1 endpoint at T _H , and reactions containing both X and Y targets plateaued at the EY2 endpoint at T _H . Endpoints EX1, EX2, EY1 and EY2 can optionally be derived from positive controls run in parallel with experimental samples.

In relation to the T _L amplification plot, if the PCR yielded an amplification curve that crossed TX and plateaued at an endpoint of EX1 above TX, this result indicated the presence of a cleaved LOCS-X associated with target X. If target Y was also present, the curve the amplification should not have changed because the cleaved LOCS-Y did not fluoresce at T _L . Therefore, the Cq values derived from the amplification curve crossing the TX allowed the target X in the sample to be quantified.

In relation to the T _H amplification plot, if PCR produced an amplification curve that crossed the TY and plateaued at EY1 or EY2, this indicated the presence of a Y target. containing only target X did not cross this threshold. In the presence of both X and Y targets, LOCS-X and LOCS-Y cleavage results in an amplification curve that crosses TY and plateaus at EY2 above EY1, which is associated with cleaved LOCS-Y in the presence of only target Y. Since EX2<TY<EY1<EY2 and EX2≠TY≠EY1≠EY2, endpoints where the amplification curves plateau may indicate the presence of target X, Y, or both. However, Cq values derived from T _H amplification curves crossing TY are dependent on the amount of both X and Y targets, and are therefore only semi-quantitative for Y target. described below (quantification methods 1 and 2).

Target Y - scoring method 1

The two amplification curves generated by cleaved LOCS-X alone at T _L 39° C. and T _H 72° C. have the same potency and Cq but plateau at different endpoints EX1 and EX2, respectively. Therefore, the fluorescence signal of the digested LOCS-X in the 72° C. amplification curve (F-LOCS-X _FAF ) can be extrapolated from the 39° C. amplification curve by applying a fluorescence correction factor (FAF). FAF is the ratio of endpoints EX1 and EX2. The total fluorescence signal in the amplification curve at 72°C (F-total) comes from the fluorescence signal resulting from both cleaved LOCS-X (F-LOCS-X _FAF ) and cleaved LOCS-Y (F-LOCS-Y) . Based on this, the amplification curve at 72°C obtained by LOCS-Y can be extrapolated as follows:

,

,

The Cq values based on the amplification curve at T _H 72°C obtained by LOCS-Y using TY are fully quantified for target Y. This formula can be used for correct quantification whether the sample contains target X, Y, or both of these targets.

Target Y - scoring method 2

In the presence of both X and Y targets, the experimentally determined Cq values at 72°C (Cq _observed ) are biased compared to the Cq values for a sample containing the same concentration of Y target without target X. Relationship between Cq _observed and expected Cq, determined by standard curve for target Y in the absence of target X at 72°C (Cq _{standard curve for target} Y) is described below:

Since the Cq _{standard curve for the Y target} and the relative shift Cq are functions of the copy number of the Y target, the copy number of the Y target can be calculated based on the experimentally determined value of Cq ( _{Cq observed} ). This relative shift of the Cq value is characterized by a mathematical (logistic) dependence on the natural logarithm of the value (number of copies of the target Y/number of copies of the target X). When determining Cq values with a threshold equal to half the maximum fluorescence level on the amplification curve measured at 72°C, the logistic dependence can be expressed by the following formula:

where y=relative shift Cq, L=maximum possible shift Cq, k=slope factor, x=ln (number of copies of target Y/number of copies of target X); note that the copy number of target X is determined from the amplification curves at 39°C.

Including a shift factor for the Cq value in the standard curve equation for a Y target determined in the absence of an X target allows the Cq value to be corrected in the presence of an X target and subsequently results in a correct quantification of the Y target.

The above strategy can be adapted to detect any targets, however, in this particular example, the X target is the CTcry gene and the Y target is the NGopa gene; amplicon accumulation of each target was monitored at low temperature (T _L 39°C) and higher temperature (T _H 72°C) during PCR for fluorescence changes associated with LOCS-X (LOCS-19) and LOCS-Y (LOCS- twenty); both LOCS were labeled with the same fluorophore (JOE).

Oligonucleotides

Oligonucleotides specific to this experiment included: 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 sequence listing. Oligonucleotides specific for amplification and quantification of CTcry were LOCS-19, partzyme A9, partzyme B9, forward primer 7 and reverse primer 7. Oligonucleotides specific for amplification and quantification of NGopa were LOCS-20, Partzyme A10, Partzyme B10, forward primer 8 and reverse primer 11.

Reaction conditions

Real-time target sequence detection was performed in a total reaction volume of 20 μl using a BioRad® CFX96 thermal cycler. The cycle parameters were: 95°C for 2 minutes, then 10 cycles of reduction, including 95°C for 5 seconds and 61°C for 30 seconds (0.5°C decrease per cycle) and 40 cycles, including 95°C for 5 seconds, 52°C for 40 seconds, 39°C for 1 s, and 72°C for 1 s (data obtained at both 39°C and 72°C steps). Each reaction mixture contained 40 nM of each forward primer, 200 nM of each reverse primer, 200 nM of each partzyme, 100 nM of LOCS-19 reporter, 200 nM of LOCS-20 reporter, and 1x PlexMastermix (Bioline). The reaction mixtures did not contain a target (NF SH0) or contained a synthetic G-Block X sequence (20000, 4000, 800, 160 or 32 copies), a synthetic G-Block sequence of the NGopa gene (20000, 4000, 800, 160 or 32 copies) , different concentrations of the synthetic G-Block sequence of the CTcry gene (20000, 4000, 800, 160 or 32 copies) against the background of the synthetic G-Block sequence of the CTcry gene (20000, 4000, 800, 160 or 32 copies) or different concentrations of the synthetic G- Block of the NGopa gene (20000, 4000, 800, 160 or 32 copies) against the background of the synthetic G-Block sequence of the CTcry gene (20000, 4000, 800, 160 or 32 copies).

results

Two MNKzymes (MNKzyme 9 and MNKzyme 10) were used to monitor the amplification of target nucleic acids during real-time PCR by cleavage of the respective LOCS reporters (LOCS-19 and LOCS-20, respectively). MNKzyme 9 was designed to detect sequences homologous to CTcry to detect Chlamydia trachomatis and to cleave and open LOCS-19. MNKzyme 10 was designed to detect sequences homologous to NGopa to detect Neisseria gonorrhoeae and to cleave and open LOCS-20.

The results shown in Figures 17A and 17B show comparative amplification curves obtained on the JOE channel using reaction mixtures containing 20,000 copies of CTcry or NGopa, or 20,000 copies of both target genes, measured at 39°C and 72°C, respectively. The results are presented as average values for reactions in duplicate. At 39°C, samples containing 20,000 copies of the CTcry template (solid black line) showed amplification curves with fluorescence levels above TX (horizontal black line, indicated as TX) and reaching EX1 (horizontal black line, indicated as EX1). At 39°C, samples containing 20,000 copies of CTcry and NGopa templates each (solid gray line) showed amplification curves with fluorescence levels exceeding TX and reaching EX1, while samples not containing CTcry templates (dashed black line) did not exceed TX. and did not reach EX1. Thus, the amplification curve at 39°C, which reached a plateau at point EX1, confirmed the presence of CTcry in the sample. The graph shows samples containing 20,000 copies of CTcry alone (solid black line) and 20,000 copies of CTcry and NGopa (solid gray line), with similar Cq values.

Since the presence of NGopa did not affect Cq values at 39°C, it can be used to quantify CTcry in a sample. Summary results of quantification of CTcry in samples of all combinations of 0, 32, 160, 800, 4000 and 20000 copies of the CTcry target gene and 0, 32, 160, 800, 4000 and 20000 copies of the NGopa target gene are shown in Table 6. NO means no Cq value determined at 39°C, and thus the absence of CTcry in the sample.

Figure 17B shows amplification curves at 72°C for samples containing 20,000 copies of the NGopa template (dashed black line) with fluorescence levels exceeding TY (black horizontal line labeled TY) and plateauing at the EY1 point (black horizontal line , designated as EY1). At 72°C, samples containing 20,000 copies of CTcry and NGopa (solid gray line) showed amplification curves with fluorescence levels exceeding TY and plateaued at the EY2 point (black horizontal line, labeled EY2), while samples that did not containing NGopa target matrices (solid black line) did not exceed TY and did not reach EY1 and EY2, and reached a plateau only at the end point EX2 (black horizontal line, designated as the end point EX2). Therefore, the amplification curve at 72° C., which plateaued at point EX2, confirmed the presence of the CTcry target, but not NGopa in the sample; plateauing at EY1 confirmed the presence of NGopa but not CTcry in the sample; the plateau at point EY2 confirmed the presence of both CTcry and NGopa in the sample.

At 72°C, the Cq value of samples containing 20,000 copies of CTcry and NGopa (solid gray line) differed from the Cq value of samples containing 20,000 copies of NGopa matrix only (dashed black line). The presence of CTcry in the sample in this example could shift the Cq value of NGopa at 72°C by about -4.77. Therefore, the use of Cq values at 72°C without normalization made it possible to determine the concentration of ^NGopa , which was underestimated by a factor of 24.77 compared to the expected value, assuming the efficiency of PCR amplification to be 100%. A summary analysis of amplification at 72°C of samples of all combinations of 0, 32, 160, 800, 4000 and 20000 copies of the CTcry target gene and 0, 32, 160, 800, 4000 and 20000 copies of the NGopa target gene without normalization is shown in Table 7 for Cq values and in Table 8 for NGopa quantification. NO means no Cq value determined at 72°C and thus no NGopa in the sample. The results show that this unnormalized method is fully quantitative for CTcry and semi-quantitative for NGopa.

The results shown in Figure 18 show comparative amplification curves obtained on the JOE channel using reaction mixtures containing all possible combinations of 0, 32 and 20,000 copies of the CTcry target gene and 0, 32 and 20,000 copies of the NGopa target gene measured at 39°C and 72°C, respectively. The results are presented as average values for reactions in duplicate. The samples presented in the graphs contained the amount of NGopa indicated on the left side of the graphs. Samples containing 20,000 copies of CTcry are represented by a dotted black line, samples containing 32 copies of CTcry are represented by a solid gray line, and samples containing 0 copies of CTcry are represented by a solid black line. From the non-normalized amplification curves obtained at 72° C. in Figures 18B and 18E, it can be seen that the presence of CTcry in the sample shifted the Cq values. The results were further analyzed using quantification method 1. Subtraction of the amplification curve at 39° C. multiplied by FAF from the amplification curve at 72° C. (since F-LOCS-Y = F-total - F-LOCS-X _FAF ) allowed obtain extrapolated amplification curves corresponding to the cleaved LOCS-20 fluorescence signal without the contribution of cleaved LOCS-19 (Figures 18C and 18F). All extrapolated amplification curves at 72° C. showed similar Cq values in the presence of the same amount of NGopa template, regardless of the amount of CTcry in the sample. Figure 181 shows that the extrapolated amplification curves at 72°C did not show significant amplification in the absence of the NGopa template in the samples. The effect of F-LOCS-X _FAF subtractive normalization is further shown in Table 9 (F-LOCS-X _FAF normalized Cq values) and Table 10 (NGopa quantification after F-LOCS-X _FAF normalization), which represent the analysis amplification of samples of all combinations of 0, 32, 160, 800, 4000 and 20000 copies of the CTcry target gene and 0, 32, 160, 800, 4000 and 20000 copies of the NGopa target gene after normalization by subtracting the F-LOCS- _{XFAF signals} . NO means no Cq value determined at 72° C. and thus no Y target in the sample.

For amplification curves based on data obtained at 72°C, the Cq values for samples containing both CTcry and NGopa were shifted relative to the sample containing only NGopa. The results were further analyzed using quantification method 2. Figure 19 shows the logistic regression of the natural logarithm of the value (NGopa copy number/CTcry copy number) versus relative Cq shift, where the Cq threshold was set at half the maximum fluorescence level in the amplification curve, following the following formula:

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

Since the number of copies of CTcry was determined based on data obtained at 39°C, the above formula can be used to determine the number of copies of NGopa in a sample based on the experimental Cq (Cq _observed ) at 72°C using a mathematical solution program. Table 11 shows observed Cq values at 72°C with relative Cq shift factors calculated for samples of all combinations of 0, 32, 160, 800, 4000 and 20000 copies of the target gene CTcry and 0, 32, 160, 800, 4000 and 20000 copies of the NGopa target gene, presented as a mean of two replicates. Table 12 shows the determination of the number of copies using the values of Cq, normalized by the coefficient of relative shift, for the same samples.

Example 13: Simultaneous detection of six targets on two fluorescence channels using six LOCS reporters

In the following example, LOCS reporters were used to increase the number of targets that could be detected on two fluorescence channels. In this example, three targets were detected on each of two fluorescent channels. In this example, all six LOCS reporters were labeled 5' with a fluorophore (JOE or Atto101) and 3' with a quencher (3IABkFQ or 3IAbRQSp, respectively). The loop regions of each of the LOCS reporters contained their own nucleic acid substrate, and the stem regions contained a series of complementary base pairs holding the LOCS reporter in a loop-stem configuration. In this configuration, the fluorophore and quencher were in close proximity, and in the absence of a target, the fluorescence was quenched.

Oligonucleotides

Oligonucleotides specific to this experiment included: LOCS-21 (SEQ ID NO: 88), LOCS-22 (SEQ ID NO: 89), LOCS-23 (SEQ ID NO: 90), LOCS-24 (SEQ ID NO: 90). 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), Forward Primer 12 (SEQ ID NO: 106), reverse primer 12 (SEQ ID NO: 107), forward primer 9 (SEQ ID NO: 54), reverse primer 9 (SEQ ID NO: 55), forward primer 13 (SEQ ID NO: 108 ), reverse primer 13 (SEQ ID NO: 109), forward primer 14 (SEQ ID NO: 110), reverse primer 2 (SEQ ID NO: 10), forward primer 1 (SEQ ID NO: 7), reverse primer 1 ( SEQ ID NO: 8), forward primer 15 (SEQ ID NO: 111), reverse primer 15 (SEQ ID NO: 112). The sequences are listed in the sequence listing. Oligonucleotides specific for gpd amplification and detection were LOCS-21, partzyme A14, partzyme B14, forward primer 12 and reverse primer 12. Oligonucleotides specific for gpd3 amplification and detection were LOCS-22, partzyme A15 , partzyme B15, forward primer 9, and reverse primer 12. Oligonucleotides specific for amplification and detection of porA were LOCS-23, partzyme A16, partzyme B16, forward primer 13, and reverse primer 13. Oligonucleotides specific for amplification and TV-Btub detection were LOCS-24, partzyme A17, partzyme B17, forward primer 14 and reverse primer 2. Oligonucleotides specific for MgPa amplification and detection were LOCS-25, partzyme A18, partzyme B18, forward primer 1 and reverse primer 1. Oligonucleotides specific for LGV amplification and detection were LOCS-26, partzyme A19, partzyme B19, forward prime ep 15 and reverse primer 15.

Reaction conditions

Real-time amplification and target sequence detection were performed in a total reaction volume of 20 μl using a BioRad® CFX96 thermal cycler. Cycle parameters were: 40°C for one second, 70°C for one second, 85°C for one second, 95°C for 2 minutes, 10 descent cycles including 95°C for 5 seconds and 61°C for 30 seconds (0.5°C decrease per cycle) and 40 cycles including 95°C for 5 seconds, 52°C for 40 seconds, and 65°C for 1 second (data taken on step 65°C), followed by 40°C for one second, 70°C for one second and 85°C for one second. Melting curve parameters ranged from 20° C. to 95° C. in 0.5° C. increments with a 5 second hold (data logging was performed during the hold step). All reactions were performed in duplicate, reaction mixtures contained 40 nM each forward primer, 200 nM each reverse primer, 200 nM each partzyme A, 200 nM each partzyme B, 200 nM each of LOCS-21, LOCS-22, LOCS-24 and LOCS-25 and 150 mM each of LOCS-23 and LOCS-26, 8 mM MgCl ₂ (Bioline), 0.2 mM dNTP (Bioline), 2 units of MyTaq polymerase (Bioline) and 1x NH4 buffer (Bioline). The reaction mixtures contained a G-Block template (10,000 copies) homologous to the gpd and/or gpd3 and/or porA and/or TV-Btub and/or MgPa and/or LGV genes, or did not contain a target (NF H ₂ O).

results

Using the in vitro target amplification method known as PCR, six MNCzymes (MNCzyme 14, MNCzyme 15, MNCzyme 16, MNCzyme 17, MNCzyme 18, and MNCzyme 19) were used to monitor the amplification of target nucleic acids in real time by cleavage of the respective LOCS reporters. (LOCS-21, LOCS-22, LOCS-23, LOCS-24, LOCS-25 and LOCS-26, respectively). The presence of any or all targets for each channel (JOE and Texas Red) was determined by the increase in fluorescence during the PCR amplification phase (data not shown); individual target recognition was then performed based on unique LOCS melting signatures. MNKzyme 14 was designed to detect sequences homologous to the gpd gene and to cleave and open LOCS-21; MNKzyme 15 was designed to detect sequences homologous to the gpd3 gene and to cleave and open LOCS-22; MNKzyme 14 was designed to detect sequences homologous to the porA gene and to cleave and open LOCS-23; MNKzyme 17 was designed to detect sequences homologous to TV-Btub and cleave and open LOCS-24; MNKzyme 18 was designed to detect sequences homologous to the MgPa gene and to cleave and open LOCS-25; and MNKzyme 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 MNCzymes, primers and LOCS oligonucleotides. The presence of gpd, gpd3, porA, TV-Btub, MgPa, LGV, or various combinations of six genes (representing a multiple infection sample) was detected by an increase in signal on one or both of the JOE and Texas Red channels (data not shown).

The results shown in Figures 20-22 show the corresponding melting curve signatures obtained after amplification in reaction mixtures containing 10,000 copies of targets gpd (Figure 20A), gpd3 (Figure 20B), porA (Figure 20C), both gpd and gpd3 (Figure 20D).), both gpd and porA (Figure 20E), both gpd3 and porA (Figure 20F), or all three targets gpd, gpd3 and porA (Figure 22A), TV-Btub (Figure 21A) , MgPa (Figure 21B), LGV (Figure 21C), both TV-Btub and MgPa (Figure 21D), both TV-Btub and LGV (Figure 21E), both MgPa and LGV (Figure 21F), or all three targets TV-Btub, MgPa and LGV (Figure 22B). Results were means of duplicate reactions plotted using Microsoft Excel (version 14).

The summary data from this example and shown in Table 13 demonstrate that the LOCS melt signatures obtained in the presence of the gpd target gene (Tm=53°C, 68°C and 85°C) differed from the LOCS melt signatures obtained in the presence of target genes gpd3 (Tm=30°C, 43°C, 77°C and 85°C) and porA (Tm=68°C and 77°C). This example demonstrates that it is possible to achieve simultaneous detection of TV-Btub, MgPa and/or LGV target genes in a second channel in addition to detection of gpd, gpd3 and/or porA. The LOCS melt signature obtained in the presence of the TV-Btub target gene (Tm=30°C, 42°C, 66°C and 82°C) differs from the LOCS melt signatures obtained in the presence of the MgPa target genes (53°C , 66°C and 82°C) and LGV (Tm=68°C). In addition, the melting signatures of LOCS in the presence of two or more target genes read on the same channel also differ from the aforementioned melting signatures obtained in the presence of only one target gene. In the presence of gpd and gpd3 target genes in the same reaction mixture, the unique signature of the LOCS melting curve (Tm=30°C, 43°C, 53°C and 85°C) on the JOE channel indicated the detection of both targets. Similarly, in the presence of gpd and porA or gpd3 and porA in the same reaction mixture, unique melting curve signatures (Tm=53°C and 68°C and Tm=30°C, 43°C, 68°C and 77°C, respectively ) indicated the detection of two targets and allowed them to be specifically identified. Similarly, the LOCS melting signatures on the Texas Red channel were unique for each combination of 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). In the presence of all gpd, gpd3 and porA target genes in one reaction mixture, the unique signature of the LOCS melting curve (Tm=30°C, 43°C, 53°C and 68°C) on the JOE channel indicated the detection of all three targets. Similarly, the presence of TV-Btub, MgPa and LGV targets in the same sample resulted in a unique LOCS melting signature (Tm=30°C, 42°C, 53°C and 68°C) on the Texas Red channel.

This example demonstrates that six targets co-amplified in one well using two fluorescence channels (with three targets detected per channel) can be distinguished based on their unique LOCS melting signatures. The example shows a simple way to detect multiple targets in one well using just two fluorescent channels. Due to different stem length and base pair composition, 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 provided unique fluorescence melting curve signatures.

Example 14: Simultaneous detection and discrimination of ten targets in one well using five fluorescent channels and ten LOCS reporters

In the following example, LOCS reporters were used to increase the number of targets that could be simultaneously detected in one well using five fluorescent channels by increasing the number of targets that could be detected on each channel. In this example, two targets were detected on each of the five fluorescent channels. In this example, all ten LOCS reporters were labeled 5' with a fluorophore (FAM, JOE, AttoRho101, Cy5 or Cy5.5) and 3' with a quencher (3IABkFQ or 3IAbRQSp). The loop regions of the LOCS reporters contained a nucleic acid substrate, and the stem regions contained a series of complementary base pairs holding the LOCS reporter in a loop-stem configuration. In this configuration, the fluorophore and quencher were in close proximity, and in the absence of a target, the fluorescence was quenched.

Oligonucleotides

Oligonucleotides specific to this experiment included: LOCS-10 (SEQ ID NO: 40), LOCS-15 (SEQ ID NO: 79), LOCS-21 (SEQ ID NO: 88), LOCS-22 (SEQ ID NO: 88). 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 BIO (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 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 p2 (SEQ ID NO: 10), forward primer 7 (SEQ ID NO: 41), reverse primer 7 (SEQ ID NO: 42), forward primer 8 (SEQ ID 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), 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.

Oligonucleotides specific for amplification and detection of CTcry were LOCS-29, partzyme A9, partzyme B9, forward primer 7 and reverse primer 7. Oligonucleotides specific for amplification and detection of NGopa were LOCS-15, partzyme A10 , partzyme B10, forward primer 8, and reverse primer 11. Oligonucleotides specific for amplification and gpd detection were LOCS-21, partzyme A14, partzyme B14, forward primer 12, and reverse primer 12. Oligonucleotides specific for amplification and detection of gpd3 were LOCS-22, partzyme A15, partzyme B15, forward primer 9 and reverse primer 9. Oligonucleotides specific for amplification and detection of porA were LOCS-27, partzyme A16, partzyme B16, forward primer 13 and reverse primer 13. Oligonucleotides specific for TV-Btub amplification and detection were LOCS-24, partzyme A17, partzyme B17, forward prime ep 14 and reverse primer 2. Oligonucleotides specific for amplification and detection of MgPa were LOCS-28, partzyme A18, partzyme B18, forward primer 1 and reverse primer 1. Oligonucleotides specific for amplification and detection of LGV were LOCS-10, Partzyme A19, Partzyme B19, Forward Primer 15 and Reverse Primer 15. Oligonucleotides specific for polA amplification and detection were LOCS-30, Partzyme A20, Partzyme B20, Forward Primer 16 and Reverse Primer 16. Oligonucleotides specific for TFRC amplification and detection were LOCS-31, partzyme A21, partzyme B21, forward primer 10 and reverse primer 10.

Reaction conditions

Real-time amplification and target sequence detection were performed in a total reaction volume of 20 μl using a BioRad® CFX96 thermal cycler. The cycle parameters were: 31°C for one second, 42°C for one second, 53°C for one second, 60°C for one second, 95°C for 2 minutes, 10 cycles of reduction, including 95°C for 5 seconds and 61°C for 30 seconds (0.5°C decrease per cycle) and 40 cycles including 95°C for 5 seconds, 52°C for 40 seconds and 65°C for 5 seconds (data obtained at the 65°C step), and then 31°C for one second, 42°C for one second, 53°C for one second and 60°C for one second. Melting curve parameters ranged from 20° C. to 95° C. in 0.5° C. increments with a 5 second hold (data logging was performed during the hold step). All reactions were performed in duplicate, the reaction mixtures contained 40 nM each forward primer, 200 nM each reverse primer, 200 nM each partzyme A, 200 nM each partzyme B, 200 nM each of reporters LOCS-10, LOCS-15, LOCS- 21, LOCS-22, LOCS-24, LOCS-27, LOCS-29 and LOCS-31 and 150 mm each of LOCS-28 and LOCS-30, 8 mM MgCh (Bioline), 0.2 mM dNTP (Bioline) , 2 units of MyTaq polymerase (Bioline) and 1x NH4 buffer (Bioline). The reaction mixtures contained a G-Block template (10,000 copies) homologous to the NGopa and/or porA, gpd and/or gpd3, TV-Btub and/or MgPa, CTcry and/or LGV or polA genes, or did not contain a target (NF H ₂ O). All reactions contained a background of 10,000 copies of human genomic DNA containing the TFRC target gene, which serves as an endogenous control. The detection of the TFRC gene was monitored in each reaction as an internal control by increasing fluorescence in the Cy5.5 channel (data not shown).

results

Using the in vitro target amplification method known as PCR, ten MNCzymes (MNCzyme 9, MNCzyme 10, and MNCzyme 14-21) were used to monitor amplification of target nucleic acids in real time by cleavage of the respective 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 both targets in each channel (FAM, HEX, Texas Red, Cy5 and Cy5.5) was determined by the increase in fluorescence in the PCR amplification phase, and then recognition of individual targets was performed by LOCS melting signatures.

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

In this experiment, amplification and detection were performed in one tube containing all partzymes for all MNCzymes, all primers and all LOCS oligonucleotides. The presence of any of the target genes NGopa, porA, gpd, gpd3, TV-Btub, MgPa, CTcry, LGV, polA, TFRC, or various combinations of these ten target genes (representing a multiple infection sample) was detected by an increase in signal on any of the channels FAM, HEX, Texas Red, Cy5 or Cy5.5 (data not shown).

The results shown in Figure 23 show the corresponding melting curve signatures obtained after amplification in reaction mixtures containing 10,000 copies of NGopa targets (Figure 23A), porA (Figure 23B), both NGopa and porA (Figure 23C), gpd (Figure 23D), gpd3 (Figure 23E), both gpd and gpd3 (Figure 23F), TV-Btub (Figure 23G), MgPa (Figure 23H), both TV-Btub and MgPa (Figure 231), CTcry (Figure 23J), LGV (figure 23K), both CTcry and LGV (figure 23L), polA (figure 23M) or TFRC (figure 23N). Results were means of duplicate reactions plotted using Microsoft Excel (version 14).

The summary of data from this example and shown in Table 14 demonstrates that the LOCS melt signatures obtained in the presence of a single target were different from the LOCS melt signatures obtained in the presence of a second target fluorescing on the same channel. In addition, the LOCS melt signature obtained in the presence of both targets on the same channel was different from the LOCS melt signature obtained in the presence of either of the two single targets detected at a particular wavelength. This result is the same for all five channels in this example. All LOCS melting signatures obtained on the FAM channel in the presence of target genes NGopa (Tm=53°С), porA (Tm=30°С and 76°С), as well as both NGopa and porA (Tm=30°С and 53°C) differed from each other. All LOCS melting signatures obtained on the HEX channel in the presence of target genes gpd (53°C and 70°C), gpd3 (30°C, 43°C and 76°C), and both gpd and gpd3 (30 °С, 43°С and 53°С) differed from each other. All LOCS melt signatures obtained on the Texas Red channel in the presence of target genes TV-Btub (31°C, 41°C, 59°C and 83°C), MgPa (63°C), as well as TV-Btub, and MgPa (31°С, 41°С and 63°С) differed from each other. All LOCS melting signatures obtained on the Cy5 channel in the presence of target genes CTcry (61°C and 79°C), LGV (47°C and 80°C), and both CTcry and LGV (47°C and 61 °C) were different from each other. In this example, human genomic DNA (TFRC) was detected on the Cy5.5 channel in all reactions; thus, it served as an endogenous control. Therefore, the reaction that detected polA simultaneously detected TFRC, as demonstrated in the LOCS melt signature by two peaks (39° C. and 59° C.) corresponding to the target genes TFRC and polA, respectively. The LOCS melt signatures obtained on the Cy5.5 channel in the presence of polA and TFRC (39°C and 59°C) differed from the LOCS melt signature obtained in the presence of TFRC (39°C and 80°C) by itself, providing a difference between reactions containing polA (polA and TFRC melting signatures) and reactions not containing polA (TFRC LOCS melting signature only).

This example demonstrates that ten target genes co-amplified in one well using five fluorescent channels (with two targets detected in each well) can be distinguished based on their unique LOCS melting signatures. The example shows a simple way to detect multiple targets in a single well using five fluorescent channels, doubling the number of targets detected on a single PCR machine.

Example 15: Methods for single-channel multiplex analysis in one well by measuring fluorescence at specified temperatures

The following example provides alternative protocols for detecting and distinguishing multiple targets at a single wavelength without the need to perform a full melt curve analysis over a wide range of temperatures, as previously illustrated in Examples 1 through 6. This example demonstrates several methods for detecting and identifying three targets at a single wavelength. wavelength using modified LOCS protocols combined with a choice of analysis strategies. Targets X, Y and Z can be detected using three X, Y and Z LOCS reporters, each of which can be labeled with the same fluorophore. Each of the three LOCS reporters contained its stem regions with different melting points, with Tm LOCS-X < Tm LOCS-Y < Tm LOCS-Z. In addition, each of the LOCS reporters contained its own loop regions containing a substrate specific for MNCzymes X, Y, or Z, respectively. MNCzymes X, Y, or Z were designed to recognize and detect X, Y, and Z targets, respectively, and cleave X, Y, and Z LOCS reporters, respectively. A limited number of fluorescence measurements (a total of six or less) could be performed before and/or after PCR amplification of the targets. Measurements at certain temperatures and at certain points in time can be used in various methods of analysis. A summary of them is given in Table 15.

The strategies described in this example can be adapted to detect any targets, however, in this particular example, target X was the TFRC gene, target Y was the rpoB gene, and target Z was the pss gene detected by the LOCS-X (LOCS-22) probes. LOCS-Y (LOCS-21) and LOCS-Z (LOCS-23), respectively, labeled with the same fluorophore (JOE). The detection temperatures used were: T _Xa =40.5°C; T _Xb =56.5°C; T _Xc =78.5°C; T _0b =32°C; T _Xb =40.5°C; T _Yb =56.5°C; T _Zb =65.5°C; T _Xc =39.5°C; T _Yc =56.5°C; T _Zc =65.5°C; T _Xd =40°C; T _Yd =51°C; and T _Zd =65°C. Then in the next analysis was assigned a value of N=0.5°C.

Analysis Protocol A1

At the first defined temperature T _Xa , LOCS-X cleavage generated a significant fluorescence signal compared to the no-matrix control above the FXa threshold. At the same temperature, the presence of cleaved LOCS-Y and/or LOCS-Z did not make a significant contribution to the production of the fluorescence signal due to the higher Tm of the stems of these LOCS and, therefore, the fluorescence level remained below the FXa threshold compared to the template-free control. Post-amplification fluorescence measurement at T _Xa allowed for the specific detection of cleaved LOCS-X.

At a second defined temperature T _Ya above T _Xa , cleaved LOCS-Y generated a relative fluorescence signal well above the FYa threshold. At T _Ya , the relative fluorescence signal generated by cleaved LOCS-X and/or LOCS-Z was significantly less than the threshold FYa. Thus, the measurement of fluorescence after amplification at T _Ya allowed for the specific detection of cleaved LOCS-Y.

At the third defined temperature T _Za , which exceeded the temperatures T _Xa and T _Ya , cleaved LOCS-Z generated a relative fluorescence signal well above the threshold value FZa. At T _Za , the relative fluorescence signal generated by cleaved LOCS-X and/or LOCS-Y was significantly less than the FZa threshold. Thus, the measurement of fluorescence after amplification at Tza allowed the specific detection of cleaved LOCS-Z. The detection of each cleaved LOCS reporter indicates the presence of the corresponding target in the sample.

Analysis Protocol A2

Instead of using thresholds, an algorithm can be used to determine split LOCS by splitting one or more LOCS in a sample. This algorithm used the ratio of fluorescence levels compared to a no-matrix control, measured at each of three detection temperatures (temperatures T _Xa , T _Ya and T _Za ). The ratio of relative fluorescence levels measured at the above three temperatures was unique for each of the possible combinations of cleaved LOCS and functioned as a fingerprint indicating the presence of the corresponding target gene in the sample. The algorithm consisted of seven logical tests; one logical test was assigned to each possible combination of split and non-split LOCS to determine the presence of one or more of the split LOCS. The possible combinations were (X only), (Y only), (Z only), (X and Y only), (X and Z only), (Y and Z only), or (X, Y and Z). In order to determine a sample as positive for a certain combination of targets, it was necessary for it to be set to "TRUE" in a particular logical test. The logic test determined whether (1) the ratio of the fluorescence signal measured at three different temperatures was within a predetermined range of values, and (2) the fluorescence signal was significantly greater than that of the no-matrix control. The sample could be set to TRUE on up to one set of logical tests that indicated a particular combination of split LOCS-X, LOCS-Y and LOCS-Z and therefore revealed the presence of the corresponding target(s). If the sample was set to "FALSE" in all logical tests, then the sample was defined as negative, containing no split LOCS, indicating the absence of all three targets in the sample.

Analysis Protocol B1

At the first determined temperature T _0b, none of the cleaved LOCS-X, LOCS-Y, or LOCS-Z generated a fluorescence signal significantly greater than that of the no-template control. At a second defined temperature T _Xb above T _0b , cleaved LOCS-X generated a significant fluorescence signal, while cleaved LOCS-Y and/or LOCS-Z did not significantly contribute to the production of the fluorescence signal due to the higher Tm of the stems of these LOCS. The difference in fluorescence signals after amplification at temperatures T _0b and T _Xb exceeded the threshold value FXb in the presence of cleaved LOCS-X and was below it in the absence of cleaved LOCS-X, and therefore, this allowed the specific detection of cleaved LOCS-X. At a third defined temperature T _Yb above T _Xb cleaved LOCS-Y generated a significant fluorescence signal. On the other hand, cleaved LOCS-Z did not significantly contribute to the production of the fluorescence signal at T _Yb due to the higher Tm of its stem. Cleaved LOCS-X did not make an additional significant contribution to the increase in fluorescence production at the temperature T _Yb compared to the temperature T _Xb . The difference in fluorescence signals after amplification at temperatures T _Xb and T _Yb exceeded the FYb threshold in the presence of cleaved LOCS-Y and was below it in the absence of cleaved LOCS-Y, and therefore, this allowed the specific detection of cleaved LOCS-Y. At the fourth defined temperature, T _Zb above T _Yb , cleaved LOCS-Z generated a significant fluorescence signal. Cleaved LOCS-X and/or LOCS-Y did not make an additional significant contribution to the increase in fluorescence production at the temperature T _Zb . The difference in fluorescence signals after amplification at temperatures T _Yb and T _Zb exceeded the FZb threshold in the presence of cleaved LOCS-Z and was below it in the absence of cleaved LOCS-Z, and therefore, this allowed specific detection of cleaved LOCS-Z.

The above calculations are described by the following formulas:

To determine the presence of cleaved LOCS-X,

if the fluorescence level after amplification at temperature T _Xb is the fluorescence level after amplification at temperature T _0b > threshold FXb, then LOCS-X is present in the sample.

To determine the presence of cleaved LOCS-Y,

if the fluorescence level after amplification at temperature T _Yb is the fluorescence level after amplification at temperature T _Xb > FYb threshold, then LOCS-Y is present in the sample.

To determine the presence of cleaved LOCS-Z,

if the fluorescence level after amplification at a temperature T _Zb is the fluorescence level after amplification at a temperature T _Yb > FZb threshold, then LOCS-Z is present in the sample.

The detection of each cleaved LOCS reporter indicates the presence of the corresponding target gene in the sample.

Analysis Protocol B2

Instead of using thresholds, an algorithm can be used to determine cleaved LOCS when one or more LOCS in a sample is cleaved, using a ratio of values calculated from post-amplification fluorescence level differences measured at T _0b , T _Xb , T _Yb , and T _Zb . The ratio of the values of (a) (fluorescence level after amplification at T _Xb - fluorescence level after amplification at T _0b ), (b) (fluorescence level after amplification at T _Yb - fluorescence level after amplification at T _Xb ) and (c ) (fluorescence level after amplification at temperature T _Zb - fluorescence level after amplification at temperature T _Yc ) was unique for each of the possible combinations of split LOCS and functioned as a fingerprint indicating the presence of the corresponding target/targets in the sample. The algorithm consisted of eight logical tests; one logical test was assigned to each possible combination of split and non-split LOCS to determine the presence of one or more of the split LOCS. The possible combinations were (no target), (X only), (Y only), (Z only), (X and Y only), (X and Z only), (Y and Z only), or (X, Y and Z). In order to determine a sample as positive for a certain combination of targets, it was necessary for it to be set to "TRUE" in the logical test. The logic test determined whether the difference ratio of the above three values was within a predetermined range of values. A sample could be set to TRUE in up to one logical test that indicated a particular combination of split LOCS-X, LOCS-Y, and LOCS-Z and was indicative of the presence of the corresponding target(s) or, when set to TRUE for a logical test (no target), indicated the absence of targets.

Analysis Protocol C1

Detection of cleaved LOCS after amplification at a specific temperature can also be performed using information derived from the difference between fluorescence measurements before and after amplification at specific temperatures. This method is an alternative to determining the relative level of fluorescence after amplification compared to a control without a template or with a reference temperature (T _0b ), as described above in protocols A and B, respectively. At the first defined temperature T _Xc , cleaved LOCS-X generated a significant fluorescence signal, and the difference between pre- and post-amplification fluorescence measurements at T _Xc exceeded the FXc threshold. On the other hand, the presence of cleaved LOCS-Y and/or LOCS-Z did not lead to the production of a significant fluorescence signal at T _Xc due to the higher stem Tm, and the difference in fluorescence measurement at T _Xc before and after amplification did not reach the threshold FXc. Thus, fluorescence measurements before and after amplification at T _Xc allowed the specific detection of cleaved LOCS-X. The rising fluorescence signal generated by split LOCS-Y at a second temperature T _Yc above T _Xc (where T _Yc >T _Xc ) was well above the FYc threshold. On the other hand, the increasing fluorescence signal generated by cleaved LOCS-X and/or LOCS-Z at T _Yc compared to T _Xc was significantly less than the FYc threshold. The increasing fluorescence signal at T _Yc compared to T _Xc was determined by calculating the difference between the fluorescence level before and after amplification at T _Yc and the difference between the fluorescence level before and after amplification at T _Xc . Thus, the information obtained from measurements of the fluorescence level before and after amplification at T _Xc and T _Yc allowed the cleaved LOCS-Y to be specifically detected. The increasing fluorescence signal generated by split LOCS-Z at the third temperature T _Zc compared to the temperature T _Yc (where T _Zc >T _Yc ) is well above the FZc threshold. On the other hand, the increasing fluorescence signal generated by cleaved LOCS-X and/or LOCS-Y at T _Zc compared to T _Yc was significantly less than the FZc threshold. The increasing fluorescence signal at T _Zc compared to T _{Yc was} determined by calculating the difference between the fluorescence level before and after amplification at T _Yc and the difference between the fluorescence level before and after amplification at T _Xc . Thus, the information obtained from measurements of the fluorescence level before and after amplification at T _Yc and T _Zc allowed the cleaved LOCS-Z to be specifically detected.

The above calculations are described below:

To determine the presence of LOCS-X,

if the fluorescence level after amplification at T _Xc is the fluorescence level before amplification at T _Xc > threshold value FXc, then LOCS-X is present in the sample.

To determine the presence of LOCS-Y,

if (fluorescence level after amplification at temperature T _Yc - fluorescence level before amplification at temperature T _Yc ) - (fluorescence level after amplification at temperature T _Xc - fluorescence level before amplification at temperature T _Xc ) > FYc threshold value, then LOCS is present in the sample -Y.

To determine the presence of LOCS-Z,

if (fluorescence level after amplification at temperature T _Zc - fluorescence level before amplification at temperature T _Zc ) - (fluorescence level after amplification at temperature T _Yc - fluorescence level before amplification at temperature T _Yc ) > threshold value FZc, then LOCS is present in the sample -Z. The detection of each cleaved LOCS reporter indicates the presence of the corresponding target in the sample. This method requires six fluorescence measurements.

Analysis Protocol C2

Instead of using thresholds, an algorithm can be used to determine cleaved LOCS by cleaving one or more LOCS in a sample using a ratio of values calculated from pre-amplification and post-amplification fluorescence level differences measured at the three detection temperatures T _Xc , T _Yc and T _Zc . The ratio of values (i) fluorescence level after amplification at temperature T _XC - fluorescence level before amplification at temperature T _Xc , (ii) (fluorescence level after amplification at temperature T _Yc - fluorescence level before amplification at temperature T _Yc ) - (fluorescence level after amplification at temperature T _Xc - fluorescence level before amplification at temperature T _Xc ) and (iii) (fluorescence level after amplification at temperature T _Zc - fluorescence level before amplification at temperature T _Zc ) - (fluorescence level after amplification at temperature T _Yc - level fluorescence prior to amplification at T _Yc ) was unique for each of the possible combinations of cleaved and uncleaved LOCS and could function as a fingerprint indicating the presence of the corresponding target gene in the sample. The algorithm consisted of eight logical tests; one logical test was assigned to each possible combination of split and non-split LOCS to determine the presence of one or more of the split LOCS. The possible combinations were (no target), (X only), (Y only), (Z only), (X and Y only), (X and Z only), (Y and Z only), or (X, Y and Z). In order to determine a sample as positive for a certain combination of targets, it was necessary for it to be set to "TRUE" in the logical test. The logical test determined whether the ratio of the differences between the values (i), (ii) and (iii) was within a predetermined range of values. A sample could be set to TRUE in up to one logical test that indicated a particular combination of split LOCS-X, LOCS-Y, and LOCS-Z and thus the sample contained the corresponding target(s) or, when set to TRUE " for a logical test (no target) contained no targets.

Analysis protocol D1

Another alternative method is to measure the height of the melting peak in the melting signature, which is equivalent to the value of dFluorescence/dTemperature at Tm of each split LOCS. However, the applicability of this method is not limited to determining the value of dFluorescence/dTemperature at Tm of the split LOCS, but also includes a temperature range that includes Tm. The value of dFluorescence/dTemperature at the indicated temperature of A°C was equivalent to a fluorescence level gradient between (A-N)°C and (A+N)°C. Mathematically, this can be expressed by the following formula:

As can be seen from the formula above, to calculate the value of dFluorescence/dTemperature at A°C, two fluorescence measurements at temperatures (A-N)°C and (A+N)°C are required. Triple analysis requires three dFluorescence/dTemperature values and therefore six post-amplification fluorescence measurements. In the presence of cleaved LOCS-X, the dFluorescence/dTemperature at T _Xd exceeded the FXd threshold, but in the absence of cleaved LOCS-X, the dFluorescence/dTemperature at T _Xd did not reach the FXd threshold. In the presence of cleaved LOCS-Y, the dFluorescence/dTemperature at T _Yd exceeded the FYd threshold, but in the absence of cleaved LOCS-Y, the dFluorescence/dTemperature at T _Yd did not reach the FYd threshold. In the presence of cleaved LOCS-Z, the dFluorescence/dTemperature at T _Zd exceeded the FZd threshold, but in the absence of cleaved LOCS-Z, the dFluorescence/dTemperature at T _Zd did not reach the FZd threshold. The detection of each cleaved LOCS reporter indicates the presence of the corresponding target gene in the sample.

D2 analysis protocol

Instead of using thresholds, an algorithm can be used to determine cleaved LOCS by cleaving one or more LOCS in a sample using the ratio of dFluorescence/dTemperature values at the three detection temperatures T _Xd , T _Yd and T _Zd . The ratio of dFluorescence/dTemperature values at the above three temperatures was unique for each of the possible combinations of split LOCS and functioned as a fingerprint indicating the presence of the corresponding target(s) in the sample. The algorithm consisted of eight logical tests; one logical test was assigned to each possible combination of split and non-split LOCS to determine the presence of one or more of the split LOCS. The possible combinations were (no target), (X only), (Y only), (Z only), (X and Y only), (X and Z only), (Y and Z only), or (X, Y and Z). In order to determine a sample as positive for a certain combination of targets, it was necessary for it to be set to "TRUE" in the logical test. A logic test determined whether the ratio of dFluorescence/dTemperature values at the above three temperatures was within a predetermined range of values. A sample could be set to TRUE in at most one logical test that indicated a particular combination of split LOCS-X, LOCS-Y and LOCS-Z and thus to the corresponding target(s) or, when set to TRUE, for a logical test (no target), for no targets.

Oligonucleotides

Oligonucleotides specific to this experiment included: LOCS-22 (SEQ ID 89), LOCS-21 (SEQ ID 88), LOCS-23 (SEQ ID 90), Partzyme A22 (SEQ ID 124), Partzyme B22 (SEQ ID 125), Partzyme A23 (SEQ ID 126), Partzyme B23 (SEQ ID 127), Partzyme A24 (SEQ ID 130), Partzyme B24 (SEQ ID 131), Forward Primer 10 (SEQ ID 72), Reverse Primer 10 (SEQ ID 73), forward primer 6 (SEQ ID 25), reverse primer 6 (SEQ ID 26), forward primer 17 (SEQ ID 128) and reverse primer 17 (SEQ ID 129). The sequences are listed in the sequence listing. Oligonucleotides specific for amplification and detection of the TFRC gene were LOCS-22, partzyme A22, partzyme B22, forward primer 10 and reverse primer 10. Oligonucleotides specific for amplification and detection of the rpoB gene were LOCS-21, Partzyme A23, Partzyme B23, Forward Primer 6, and Reverse Primer 6. Oligonucleotides specific for amplification and detection of the pss gene were LOCS-23, Partzyme A24, Partzyme B24, Forward Primer 17, and Reverse Primer 17.

Reaction conditions

Real-time target sequence detection was performed in a total reaction volume of 20 μl using a BioRad® CFX96 thermal cycler. Cycle parameters were: 39.5°C for 30 seconds with data logging, 56.5°C for 30 seconds with data logging, 65.5°C for 30 seconds with data logging, 95°C for 2 minutes, 10 reduction cycles of 95°C for 5 seconds and 61°C for 30 seconds (0.5°C decrease per cycle) and 40 cycles of 95°C for 5 seconds and 52°C for for 40 seconds (data acquired at the 52°C step) and then 39.5°C for 30 seconds with data logging, 40.5°C for 30 seconds with data logging, 50.5°C for 30 seconds with data logging, 51.5°C for 30 seconds with data logging, 56.5°C for 30 seconds with data logging, 64.5°C for 30 seconds with data logging, 65.5°C for 30 seconds with data logging and 78.5°C for 30 seconds with data logging. Each reaction mixture contained 40 nM each forward primer, 200 nM each reverse primer, 200 nM each partzyme, 80 nM LOCS-22 reporter, 210 nM LOCS-21 reporter, 300 nM LOCS-23 reporter, 2 mM MgCl ₂ (Bioline) and 1x SensiFAST Probe No-ROX mix (Bioline). Reaction mixtures contained genomic DNA or G-Block synthetic target gene template (10,000 copies per target gene in NF H ₂ O) or no target (NF H ₂ O).

results

Three MNCzymes (MNCzyme 22, MNCzyme 23 and MNCzyme 24) were used during PCR to monitor the amplification of target nucleic acids in real time by cleavage of the respective LOCS reporters (LOCS-22, LOCS-21, and LOCS-23, respectively). MNKzyme 22 was designed to detect TFRC (Homo sapiens) gene sequences and cleave LOCS-22. MNKzyme 23 was designed to detect rpoB gene sequences (Klebsiella pneumoniae) and cleave LOCS-21. MNKzyme 24 was designed to detect pss (Streptococcus pneumoniae) gene sequences and cleave LOCS-23. The presence of the target gene TFRC, rpoB or pss was determined by the fluorescence signal on the JOE channel, measured at several temperatures, at the stage of PCR cycles before or after amplification.

The results shown in Figure 24 show a comparative analysis of fluorescence levels after amplification in the JOE channel using reaction mixtures containing 10,000 copies of TFRC, rpoB, pss, or all combinations of two or more target genes, measured at 40.5°C, 56, 5°C and 78.5°C. Results are the mean fluorescence levels for triplicate reactions minus the mean of triplicate reactions for the template-free control. At 40.5° C., the TFRC-containing samples exhibited a higher level of fluorescence (compared to the no-matrix control) than the FXa threshold, but this did not apply to the TFRC-free samples. At 56.5° C., samples containing rpoB had a higher level of fluorescence (compared to the control without a template) than the FYa threshold, but this did not apply to samples not containing rpoB. At 78.5° C., samples containing pss exhibited higher fluorescence (compared to the no-matrix control) than the FZa threshold, but this did not apply to samples not containing pss.

Table 16 shows the results obtained after processing the post-amplification fluorescence level (measured at 40.5°C, 56.5°C, and 78.5°C for each of the samples containing 10,000 copies of TFRC, rpoB, pss, or all combinations of the two or more target genes), using seven logical tests. Each of the seven logical tests included three criteria (second row of Table 16) that determined whether the sample contained (TFRC only), (rpoB only), (pss only), (TFRC and rpoB only), (TFRC and pss only), (rpoB and pss only) or (TFRC, rpoB and pss). The first two criteria determined whether the ratio of the two fluorescence levels measured at different temperatures (40.5°C, 56.5°C, or 78.5°C) minus the average fluorescence level for the no-matrix control at the same temperatures, greater or lesser than a predetermined value, or whether it falls within a predetermined range. The latter criterion determined whether the observed fluorescence signal was significantly higher than that of the no-matrix control. Each sample was considered to be TRUE for the logic test only if it was TRUE for all three logic test criteria. The experiment was performed using eight types of samples in duplicate, representing all combinations of 0 or 10,000 copies of the three target templates (no template control contained 0 copies of all three templates). All samples tested were set to TRUE for a maximum of one logical test, and FALSE for all other logical tests, to accurately determine the presence of the target gene. Control samples that did not contain templates were characterized by the value "FALSE" for all logical tests.

Figure 25 shows a comparison of differential fluorescence levels after amplification in the JOE channel using reaction mixtures containing 10,000 copies of TFRC, rpoB, pss, or all combinations of two or more target genes, measured at 32°C, 40.5°C, 56, 5°C. and 65.5°C in triplicate. The difference between the levels of fluorescence measured at 32°C and 40.5°C, indicated as (a), exceeded the threshold value FXb only in samples containing TFRC. The difference between fluorescence levels measured at 40.5°C and 56.5°C, indicated as (b), exceeded the FYb threshold value only in samples containing rpoB. The difference between the fluorescence levels measured at 56.5°C and 65.5°C, denoted as (c), exceeded the FZb threshold value only in samples containing pss.

Table 17 shows the results obtained after processing the differential values of post-amplification fluorescence measured at 40.5°C, 56.5°C, and 65.5°C for each of the samples containing 10,000 copies of TFRC, rpoB, pss, or all combinations of two or more target genes using eight logical tests. These differential values were calculated as the difference between the levels of fluorescence measured at 32°C and 39.5°C; 40.5°C and 56.5°C; and 56.5°C and 65.5°C. Each of the eight logical tests included two criteria (second row of Table 17) that determined whether the sample contained (no target), (TFRC only), (rpoB only), (pss only), (TFRC and rpoB only), (only TFRC and pss), (rpoB and pss only) or (TFRC, rpoB and pss). These two criteria determined whether the ratio of two differential fluorescence values was greater or lesser than a predetermined value, or whether it fell within a predetermined range. All samples tested were set to TRUE for only one logical test, and FALSE for all other logical tests, to accurately determine the presence of the target gene.

The results shown in Figure 26 show a comparison of fluorescence levels before and after amplification on the JOE channel using reaction mixtures containing 10,000 copies of TFRC, rpoB, pss, or all combinations of two or more target genes, measured at 39.5°C, 56.5°C and 65.5°C. The results are averages obtained in triplicate reactions for the values of (i) fluorescence level after amplification at 39.5°C - fluorescence level before amplification at 39.5°C, (ii) (fluorescence level after amplification at 56.5 °C - fluorescence level before amplification at 56.5°C) - (fluorescence level after amplification at 39.5°C - fluorescence level before amplification at 39.5°C) and (iii) (fluorescence level after amplification at temperature 65.5°C - fluorescence level before amplification at a temperature of 65.5°C) - (fluorescence level after amplification at a temperature of 56.5°C - fluorescence level before amplification at a temperature of 56.5°C). For samples containing TFRC, normalized fluorescence values (i) exceeded the threshold value FXc, but this did not apply to samples not containing TFRC. For samples containing rpoB, the normalized fluorescence values (ii) exceeded the FYc threshold, but this did not apply to samples not containing rpoB. For samples containing pss, the normalized fluorescence values (iii) exceeded the FZc threshold, but this did not apply to samples not containing pss.

Table 18 shows the results obtained after performing eight logical tests for the above values (i), (ii) and (iii). Each of the eight logical tests included two criteria (second row of Table 18) that determined whether the sample contained (no target), (TFRC only), (rpoB only), (pss only), (TFRC and rpoB only), (only TFRC and pss), (rpoB and pss only) or (TFRC, rpoB and pss). Each of these two criteria determined whether the ratio of two of the values (i), (ii) and (iii) was greater or less than a predetermined value, or fell within a predetermined range. All samples tested were set to TRUE for only one logical test, and FALSE for all other logical tests, to accurately determine the presence of the target gene.

The figure 27 shows a comparison of the average values of dFluorescence/dTemperature after amplification obtained on the JOE channel using reaction mixtures containing 10,000 copies of TFRC, rpoB, pss or all combinations of two or more target genes at 37°C, 50°C and 65 °C. These values were calculated from post-amplification fluorescence measurements performed at 36.5°C, 37.5°C, 49.5°C, 50.5°C, 64.5°C and 65.5°C. The value of dFluorescence/dTemperature at 37°C exceeded the threshold value FXd only for samples containing TFRC. The value of dFluorescence/dTemperature at 50°C exceeded the FYd threshold value only for samples containing rpoB. The value of dfluorescence/dTemperature at 65°С exceeded the threshold value FZd only for samples containing pss.

Table 19 shows the results obtained after processing the dFluorescence/dTemperature values measured at 37°C, 50°C, and 65.5°C for each of the samples containing 10,000 copies of TFRC, rpoB, pss, or all combinations of two or more genes - targets using eight logic tests. These values were calculated from post-amplification fluorescence measurements performed at 36.5°C, 37.5°C, 49.5°C, 50.5°C, 64.5°C and 65.5°C. Each of the eight logical tests included two criteria (second row of Table 19) that determined whether the sample contained (no target), (TFRC only), (rpoB only), (pss only), (TFRC and rpoB only), (only TFRC and pss), (rpoB and pss only) or (TFRC, rpoB and pss). These two criteria determined whether the ratio of the two values c1Fluorescence/c1Temperature was greater or less than a predetermined value, or fell within a predetermined range. All samples tested were set to TRUE for only one logical test, and FALSE for all other logical tests, to accurately determine the presence of the target gene.

Claims (237)

1. A method for determining the presence or absence of the first and second targets in the sample, including: (a) obtaining a reaction mixture by bringing the sample or its derivative, presumably containing the first and/or second targets or their amplicons, into contact with: the first and second closed stem-loop oligonucleotides, each of the closed stem-loop oligonucleotides containing a double-stranded stem fragment formed by hybridized nucleotides and connected to a closed single-stranded loop fragment formed by unhybridized nucleotides, wherein: the number of hybridized nucleotides and/or the sequence of hybridized nucleotides in the stem fragment of the first and second closed stem-loop oligonucleotides are different, each of the double-stranded stem fragments contains a fluorophore molecule attached to one strand and a quencher molecule attached to the opposite strand; and enzymes capable of cleaving or destroying single-stranded loop fragments of the first and second closed stem-loop oligonucleotides only upon contact with the target or its amplicon; (b) working up the reaction mixture: - under conditions suitable for the enzymes to induce cleavage or destruction of the loop fragment of the first and second closed stem-loop oligonucleotides, thereby forming the first and second open stem-loop oligonucleotides; and detecting the presence or absence of said first and second disclosed stem-loop oligonucleotides by treating the reaction mixture or a derivative thereof: at or above the first temperature at which the double-stranded stem fragment chains of the first disclosed stem-loop oligonucleotide dissociate, thereby facilitating the spatial separation of the fluorophore and quencher molecules of the stem fragment of the first disclosed stem-loop oligonucleotide and providing the first detectable fluorescent signal, and at or above a second temperature at which the double-stranded stem fragment chains of the second disclosed stem-loop oligonucleotide dissociate, thereby facilitating the spatial separation of the fluorophore and quencher molecules of the stem fragment of the second disclosed stem-loop oligonucleotide and providing a second detectable fluorescent signal; and: the first temperature is different from the second temperature, and the fluorophore of the first disclosed stem-loop oligonucleotide and the fluorophore of the second disclosed stem-loop oligonucleotide emit in the same color region of the visible spectrum; and (c) detecting levels of said first and second fluorescent signals at one or more temperatures, including or consisting of a temperature equal to or greater than the second temperature, which determines the presence or absence of said targets in the sample. 2. The method according to p. 1, characterized in that the enzymes include multicomponent nucleozymes (MNCzymes), and said processing of the reaction mixture includes processing the reaction mixture under conditions suitable for: binding a first multicomponent nucleo- zyme (MNKzyme) to a first target or amplicon thereof; and hybridizing the substrate strands of said first MNKzyme with a loop fragment of the first closed stem-loop oligonucleotide, thereby facilitating said cleavage of the loop fragment of the first closed stem-loop oligonucleotide by the first MNKzyme to form the first open stem-loop oligonucleotide. 3. The method according to claim 2, characterized in that the first target is a nucleotide sequence or its amplicon capable of hybridizing with the sensory strands of the first MNKzyme, thereby facilitating the assembly of the first MNKzyme. 4. The method according to p. 1, characterized in that: - the first target is an analyte, protein, compound or molecule; - enzymes include enzymes with an aptamer capable of binding to the first target; and binding of the first target to the aptamer is capable of making enzymes with the aptamer catalytically active. 5. The method according to claim 4, characterized in that the aptamer enzymes include any one or more of: apta-DNAzymes, apta-ribozymes, apta-MNKzymes. 6. The method according to any one of paragraphs. 2, 4 or 5, characterized in that: - the first target is an analyte, protein, compound or molecule; - the reaction mixture additionally contains an oligonucleotide sequence capable of hybridizing with the sensor chains of the first MNKzyme, thereby facilitating the assembly of the first MNKzyme; the first MNKzyme contains an aptamer sequence capable of binding to the first target; and - binding of the target to the aptamer can make the first MNKzyme catalytically active. 7. The method according to any one of paragraphs. 1-6, characterized in that said closed stem-loop oligonucleotides do not hybridize to said target or its amplicon during said cleavage or degradation by enzymes. 8. The method according to any one of paragraphs. 1-6, characterized in that the enzymes include restriction endonucleases, and said processing of the reaction mixture includes: processing the reaction mixture under conditions suitable for hybridizing the first target or its amplicon with the loop fragment of the first closed stem-loop oligonucleotide to form a double-stranded sequence for binding the first restriction endonuclease, which facilitates said cleavage of the loop fragment of the first closed stem-loop oligonucleotide to form the first disclosed stem-loop oligonucleotide. 9. The method of claim 8, wherein the restriction endonuclease is a nicking endonuclease capable of binding to and cleaving the loop chain of said double-stranded sequence for the first restriction endonuclease. 10. The method according to any one of paragraphs. 1-6, characterized in that the enzymes have exonuclease activity, and said processing of the reaction mixture includes: processing the reaction mixture under conditions suitable for: - hybridization of the first target or its amplicon with the loop fragment of the first closed stem-loop oligonucleotide to form the first double-stranded sequence containing the first target or its amplicon, - hybridization of the first primer-oligonucleotide with the first target or its amplicon with the formation of the second double-stranded sequence located upstream (in the direction of 5') relative to the first double-stranded sequence containing the first target or its amplicon, association of the first enzyme having exonuclease activity with the loop fragment of the first closed stem-loop oligonucleotide at or near the end of the first primer-oligonucleotide, and - catalytic activity of the first enzyme having exonuclease activity, which facilitates the destruction of the loop fragment of the first double-stranded sequence containing the first target or amplicon, and the formation of the first open stem-loop oligonucleotide. 11. The method according to any one of paragraphs. 1-6, characterized in that the enzymes have exonuclease activity, and said processing of the reaction mixture includes: processing the reaction mixture under conditions suitable for: - hybridization of the first target or its amplicon with the loop fragment of the first closed stem-loop oligonucleotide to form the first double-stranded sequence containing the first target or its amplicon, association of the first enzyme having exonuclease activity with the first double-stranded sequence containing the first target or its amplicon, and - catalytic activity of the first enzyme having exonuclease activity, which facilitates the destruction of the loop fragment of the first double-stranded sequence containing the first target or amplicon, and the formation of the first open stem-loop oligonucleotide. 12. The method according to any one of paragraphs. 1-11, characterized in that the enzymes include DNAzymes and/or ribozymes requiring a first cofactor for the manifestation of catalytic activity, and said processing of the reaction mixture includes processing the reaction mixture under conditions suitable for: - linking said first cofactor to a DNAzyme and/or linking said first cofactor to a ribozyme conferring catalytic activity on the DNAzyme and/or ribozyme, - hybridization of the DNAzyme and/or ribozyme with the loop fragment of the first closed stem-loop oligonucleotide, - the catalytic activity of the DNAzyme and/or ribozyme to facilitate cleavage of the loop fragment of the first closed stem-loop oligonucleotide and the formation of the first open stem-loop oligonucleotide, and: the first target is the first cofactor. 13. The method according to p. 12, characterized in that the first cofactor is a metal ion. 14. The method according to any one of paragraphs. 1-13, characterized in that said processing further comprises processing the reaction mixture under conditions suitable for any one or more of: - binding of the second MNKzyme with the second target or its amplicon and hybridization of the substrate chains of the specified second MNKzyme with the loop fragment of the second closed stem-loop oligonucleotide, which facilitates the specified cleavage of the loop fragment of the second closed stem-loop oligonucleotide by the second MNKzyme with the formation of the second a disclosed stem-loop oligonucleotide; - hybridization of the second target or its amplicon with the loop fragment of the second closed stem-loop oligonucleotide to form a double-stranded sequence for binding the second restriction endonuclease to associate with the second double-stranded sequence, which facilitates said cleavage of the loop fragment of the second closed stem-loop oligonucleotide " to form a second open stem-loop oligonucleotide, - hybridization of the second target or its amplicon with the loop fragment of the second closed stem-loop oligonucleotide to form a second double-stranded sequence containing the second target or its amplicon, hybridization of the second primer-oligonucleotide with the second target or its amplicon to form a second double-stranded sequence located upstream (in the 5' direction) relative to the second double-stranded sequence containing the second target or its amplicon, association of the second enzyme having exonuclease activity with the loop fragment of the second closed stem-loop oligonucleotide at or near the end of the second primer-oligonucleotide, and catalytic activity of a second enzyme having exonuclease activity, which facilitates the destruction of the loop fragment of the second double-stranded sequence containing the second target or amplicon, and the formation of the second open stem-loop oligonucleotide; - hybridization of the second target or its amplicon with the loop fragment of the second closed stem-loop oligonucleotide to form a second double-stranded sequence containing the second target or its amplicon, association of a second enzyme having exonuclease activity with a second double-stranded sequence containing a second target or its amplicon, and catalytic activity of a second enzyme having exonuclease activity, which facilitates the destruction of the loop fragment of the second double-stranded sequence containing the second target or amplicon, and the formation of the second open stem-loop oligonucleotide; - binding of the second cofactor to the DNAzyme and/or binding of the second cofactor to the ribozyme conferring catalytic activity on the DNAzyme and/or ribozyme, hybridization of the DNAzyme and/or ribozyme with the loop fragment of the second closed stem-loop oligonucleotide, catalytic activity of the DNAzyme and/or ribozyme to facilitate cleavage of the loop fragment of the second closed stem-loop oligonucleotide and the formation of the second open stem-loop oligonucleotide, and: the second target is the second cofactor. 15. The method according to p. 14, characterized in that the second cofactor is a metal ion. 16. The method of claim 14, wherein the second restriction endonuclease is a nicking endonuclease capable of associating with and cleaving the loop strand of said double-stranded sequence containing the second target or its amplicon. 17. The method of claim 16, wherein the first and second restriction endonucleases are different types of restriction endonucleases. 18. The method according to claim 16, wherein the first and second restriction endonucleases are restriction endonucleases of the same type. 19. The method of claim 14, wherein the second target is a nucleotide sequence or amplicon thereof capable of hybridizing with the sensory strands of the second MNKzyme, thereby facilitating assembly of the second MNKzyme. 20. The method according to claim 14, characterized in that: - the second target is an analyte, protein, compound or molecule; - enzymes include enzymes with an aptamer capable of binding to a second target; and binding of the second target to the aptamer is capable of making enzymes with the aptamer catalytically active. 21. The method of claim 20, wherein the aptamer enzymes comprise any one or more of: apta-DNA zymes, apta-ribozymes, apta-MNK zymes. 22. The method according to paragraphs. 14, 20 or 21, characterized in that: - the second target is an analyte, protein, compound or molecule; - the reaction mixture additionally contains an oligonucleotide sequence capable of hybridizing with the sensor chains of the second MNKzyme, thereby facilitating the assembly of the second MNKzyme; the second MNKzyme contains an aptamer sequence capable of binding to the second target; and - binding of the second target to the aptamer sequence of the second MNKzyme is able to make the second MNKzyme catalytically active, facilitating the removal of the inhibitory molecule associated with the aptamer of the second MNKzyme. 23. The method according to any one of paragraphs. 1-22, characterized in that 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. 24. The method according to any one of paragraphs. 14-23, characterized in that: - the reaction mixture contains the specified first and second MNCzymes; and - the sequence of the loop fragment of the first closed stem-loop oligonucleotide capable of hybridizing with the substrate chains of the first MNCzyme differs from the sequence of the loop fragment of the second closed stem-loop oligonucleotide capable of hybridizing with the substrate chains of the second MNCzyme. 25. The method according to any one of paragraphs. 1-24, characterized in that the fluorophore of the first closed and open stem-loop oligonucleotides and the fluorophore of the second closed and open stem-loop oligonucleotides are detected on one fluorescent emission channel of the device. 26. The method according to any one of paragraphs. 1-25, characterized in that these enzymes do not induce cleavage or destruction of any of the specified target or its amplicon. 27. The method according to any one of paragraphs. 1-26, characterized in that each of the first and second closed stem-loop oligonucleotides consists of the specified double-stranded stem fragment and the specified single-stranded loop fragment. 28. The method according to any one of paragraphs. 1-27, further comprising determining the presence or absence of the third target or its amplicon in the sample by: (d) bringing the reaction mixture containing the sample or its derivative into contact with: a third closed stem-loop oligonucleotide, wherein said third closed stem-loop oligonucleotide contains a double-stranded stem fragment of hybridized nucleotides connected to a loop fragment of a closed single-stranded oligonucleotide consisting of unhybridized nucleotides, wherein: the number of hybridized nucleotides and/or the sequence of hybridized nucleotides in the stem fragment of the third closed stem-loop oligonucleotide is different from the number and sequence of hybridized nucleotides in the stem fragments of the first and second closed stem-loop oligonucleotides; the double-stranded stem fragment contains a fluorophore molecule linked to one strand and a quencher molecule linked to the opposite strand; and enzymes capable of cleaving or destroying the single-stranded loop fragment of the third closed stem-loop oligonucleotide only when it is in contact with the target or its amplicon; (e) working up the reaction mixture: - under conditions suitable for inducing cleavage or destruction of the loop fragment of the third closed stem-loop oligonucleotide by enzymes, which allows to obtain a third open stem-loop oligonucleotide; - at a third temperature at which or above which the chains of the double-stranded stem fragment of the third disclosed stem-loop oligonucleotide dissociate, thereby facilitating the spatial separation of the fluorophore and quencher molecules of the stem fragment of the third disclosed stem-loop oligonucleotide and providing a third detectable fluorescent signal; and: the third temperature is higher than the first and second temperatures, and (f) detecting levels of said first, second, and third fluorescent signals at one or more temperatures, including or consisting of a temperature equal to or greater than the third temperature, to determine the presence or absence of said targets in the sample. 29. The method according to any one of paragraphs. 1-27, further comprising determining the presence or absence of the third target or its amplicon in the sample by: (d) bringing the reaction mixture containing the sample or its derivative into contact with: a third closed stem-loop oligonucleotide, wherein said third closed stem-loop oligonucleotide contains a double-stranded stem fragment of hybridized nucleotides connected to a loop fragment of a closed single-stranded oligonucleotide consisting of unhybridized nucleotides, wherein: the number of hybridized nucleotides and/or the sequence of hybridized nucleotides in the stem fragment of the third closed stem-loop oligonucleotide matches the number and sequence of hybridized nucleotides in the stem fragments of the first or second closed stem-loop oligonucleotides; the double-stranded stem fragment contains a fluorophore molecule connected to one strand and a quencher molecule connected to the opposite strand, and the fluorophore molecule connected to the third disclosed stem-loop oligonucleotide emits in a different region of the visible spectrum compared to the fluorophore of the first and/or or a second closed stem-loop oligonucleotide; and enzymes capable of cleaving or destroying the single-stranded loop fragment of the third closed stem-loop oligonucleotide only when it is in contact with the target or its amplicon; (e) working up the reaction mixture: - under conditions suitable for inducing cleavage or destruction of the loop fragment of the third closed stem-loop oligonucleotide by enzymes, which allows to obtain a third open stem-loop oligonucleotide; at a third temperature at which the double-stranded stem fragment of the third disclosed stem-loop oligonucleotide dissociates, thereby facilitating the spatial separation of the fluorophore and quencher molecules of the stem fragment of the third disclosed stem-loop oligonucleotide and providing a third detectable fluorescent signal; (f) detecting levels of said first, second, and third fluorescent signals at one or more temperatures, including a temperature equal to or greater than the third temperature, to determine the presence or absence of said targets in the sample. 30. The method according to claim 29, characterized in that the third temperature differs from the first and/or second temperatures. 31. The method according to any one of paragraphs. 28-30, characterized in that said method comprises treating the reaction mixture under conditions suitable for any one or more of: - binding of the third MNKzyme with the third target or its amplicon and hybridization of the substrate chains of the specified third MNKzyme with the loop fragment of the third closed stem-loop oligonucleotide, which facilitates the specified cleavage of the loop fragment of the third closed stem-loop oligonucleotide by the third MNKzyme with the formation of the third a disclosed stem-loop oligonucleotide; - hybridization of the third target or its amplicon with the loop fragment of the third closed stem-loop oligonucleotide to form a double-stranded sequence for binding the third restriction endonuclease to associate with the second double-stranded sequence, which facilitates said cleavage of the loop fragment of the third closed stem-loop oligonucleotide "to form a third disclosed stem-loop oligonucleotide; - hybridization of the third target or its amplicon with the loop fragment of the third closed stem-loop oligonucleotide to form a third double-stranded sequence containing the third target or its amplicon, hybridization of the third primer-oligonucleotide with the third target or its amplicon to form a third double-stranded sequence located upstream in the 5' direction relative to the third double-stranded sequence containing the third target or its amplicon, association of the third enzyme with exonuclease activity with the loop fragment of the third closed type oligonucleotide stem-loop at or near the end of the third oligonucleotide primer, and catalytic activity of a third enzyme having exonuclease activity, which facilitates the destruction of the loop fragment of the third double-stranded sequence containing the third target or amplicon, and the formation of the third open stem-loop oligonucleotide; - hybridization of the third target or its amplicon with the loop fragment of the third closed stem-loop oligonucleotide to form a third double-stranded sequence containing the third target or its amplicon, association of a third enzyme having exonuclease activity with a third double-stranded sequence containing a third target or its amplicon, and catalytic activity of a third enzyme having exonuclease activity, which facilitates the destruction of the loop fragment of the third double-stranded sequence containing the third target or amplicon, and the formation of the third open stem-loop oligonucleotide; - binding of the third cofactor to the DNAzyme and/or binding of the third cofactor to the ribozyme, which imparts catalytic activity to the DNAzyme and/or ribozyme, hybridization of the DNAzyme and/or ribozyme with the loop fragment of the third closed stem-loop oligonucleotide, catalytic activity of the DNAzyme and/or ribozyme to facilitate cleavage of the loop fragment of the third closed stem-loop oligonucleotide and the formation of the third open stem-loop oligonucleotide, and: the third target is the third cofactor. 32. The method according to p. 31, characterized in that the cofactor is a metal ion. 33. The method according to claim 31, wherein the third restriction endonuclease is a nicking endonuclease capable of associating with and cleaving the loop chain of said double-stranded sequence containing the third target or its amplicon. 34. The method of claim 31, wherein the third target is a nucleotide sequence or amplicon thereof capable of hybridizing with the sensory strands of the third MNKzyme, thereby facilitating assembly of the third MNKzyme. 35. The method according to p. 31, characterized in that: - the target is an analyte, protein, compound or molecule; - enzymes include enzymes with an aptamer capable of binding to a third target; and - binding of the third target to the aptamer is capable of making enzymes with the aptamer catalytically active. 36. The method of claim 35, wherein the aptamer enzymes comprise any one or more of: apta-DNAzymes, apta-ribozymes, apta-MNKzymes. 37. The method according to p. 31, characterized in that: - the third target is an analyte, protein, compound or molecule; - the reaction mixture additionally contains an oligonucleotide sequence capable of hybridizing with the sensor chains of the third MNKzyme, thereby facilitating the assembly of the third MNKzyme; the third MNKzyme contains an aptamer sequence capable of binding to a third target; and binding of the third target to the aptamer sequence of the third MNKzyme is capable of making the third MNKzyme catalytically active, facilitating the removal of the inhibitory molecule associated with the aptamer of the third MNKzyme. 38. The method according to any one of paragraphs. 31 or 33-37, characterized in that: the reaction mixture contains said third MNKzyme and one or both of said first and second MNKzymes; and - the sequence of the loop fragment of the third closed stem-loop oligonucleotide capable of hybridizing with the substrate chains of the third MNKzyme differs from: sequences of the loop fragment of the first closed stem-loop oligonucleotide capable of hybridizing with the substrate chains of the first MNKzyme, and/or sequences of the loop fragment of the second closed stem-loop oligonucleotide capable of hybridizing with the substrate chains of the first MNCzyme. 39. The method according to any one of paragraphs. 28 or 31-38, characterized in that: - the number of hybridized nucleotides and/or the sequence of hybridized nucleotides in the stem fragment of the third closed stem-loop oligonucleotide differs from the number and sequence of hybridized nucleotides in the stem fragments of the first and second closed stem-loop oligonucleotides; - the third temperature is different from the first and second temperatures; and - the fluorophore of the third closed and open stem-loop oligonucleotide emits in the same color region of the visible spectrum as the fluorophores of the first and second closed and open stem-loop oligonucleotides. 40. The method according to any one of paragraphs. 28 or 31-39, characterized in that 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. 41. The method according to p. 40, characterized in that the fluorophore of the third closed and open stem-loop oligonucleotides and the fluorophore of the first closed and open oligonucleotides and / or the fluorophore of the second closed and open stem-loop oligonucleotides are detected on one and the same fluorescence emission channel of the device. 42. The method according to any one of paragraphs. 28-38 or 39, characterized in that: - the fluorophore of the first closed and open stem-loop oligonucleotide emits in the same color region of the visible spectrum as the fluorophore of the second closed and open stem-loop oligonucleotide; and - the fluorophore of the third closed and open stem-loop oligonucleotide emits in the color region of the visible spectrum, which differs from the emission region of the fluorophore of the first and second closed and open stem-loop oligonucleotides. 43. The method according to any one of paragraphs. 28-42, characterized in that 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°, 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. 44. The method according to any one of paragraphs. 28-43, characterized in that the detection of the third fluorescent signal at the third temperature indicates the presence of the third target in the sample, and the impossibility of detecting the third fluorescent signal at the third temperature indicates the absence of the third target in the sample. 45. The method according to any one of paragraphs. 1-44, characterized in that: (i) determining the presence of the first fluorescent signal at a first temperature indicates the presence of the first target in the sample, and determining the absence of the first fluorescent signal indicates the absence of the first target in the sample; and (ii) determining the presence of a second fluorescent signal at a second temperature indicates the presence of a second target in the sample, and determining the absence of a second fluorescent signal indicates the absence of a second target in the sample. 46. The method according to any one of paragraphs. 1-44, characterized in that said determination of the presence or absence of the first and second targets includes an analysis of the melting curve using said first and second fluorescent signals. 47. The method according to any one of paragraphs. 1-44, characterized in that part (c) includes detecting the levels of the specified first and second fluorescent signals when: - a temperature equal to or greater than the second temperature; and - a temperature equal to or greater than the first temperature and not reaching the second temperature. 48. The method of claim 47, wherein part (c) further comprises detecting levels of said first and second fluorescent signals at a temperature below the second temperature. 49. The method according to claim 47 or 48, wherein part (c) comprises detecting the levels of said first and second fluorescent signals during and/or after completion of the nucleic acid amplification reaction. 50. The method according to any one of paragraphs. 47-49 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. 51. The method according to any one of paragraphs. 47-50, further comprising obtaining a fluorescent signal of a positive control of the first target by repeating said method on a separate control sample containing said first target. 52. The method of claim. 51, characterized in that the control sample containing the first target contains the first target in a known concentration. 53. The method according to p. 51 or 52, characterized in that the control sample containing the first target, additionally contains the specified second target. 54. The method according to any one of paragraphs. 46-53, further comprising obtaining a second target positive control fluorescent signal by repeating said method on a separate control sample containing said second target. 55. The method of claim 54, wherein the control sample containing the second target contains the second target at a known concentration. 56. The method according to p. 54 or 55, characterized in that the control sample additionally contains the specified first target. 57. The method according to any one of paragraphs. 46-53, further comprising obtaining the fluorescent signal of the pooled positive control by repeating said method on a separate control sample containing said first and said second targets. 58. The method of claim 57 wherein the combined control sample contains the first target at a known concentration and/or the second target at a known concentration. 59. The method according to any one of paragraphs. 50-58 further comprising normalizing said first fluorescent signal and/or said second fluorescent signal using any of said positive control fluorescent signal. 60. The method according to any one of paragraphs. 46-59, further comprising obtaining a negative control fluorescent signal by repeating the method according to any one of paragraphs. 1-3 on a single negative control that does not contain: (i) said first target; or (ii) said second target; or (iii) said first target or said second target. 61. The method of claim 60, further comprising normalizing said first fluorescent signal and/or said second fluorescent signal using said negative control fluorescent signal. 62. The method according to any one of paragraphs. 46-61, further comprising comparing said first and/or second fluorescent signals with a threshold value, wherein: - the threshold value is generated using fluorescent signals obtained from a series of samples tested in accordance with the method according to any one of paragraphs. 1-3, and containing any one or more of: (i) no template control and first target, (ii) a no-matrix control and a second target, (iii) no template control, first target and second target, which allows you to determine the specified presence or absence of the first and second targets in the sample. 63. The method of claim 62, wherein a series of samples is tested using said first closed stem-loop oligonucleotide at a known concentration and/or said second closed stem-loop oligonucleotide at a known concentration. 64. The method according to any one of paragraphs. 1-63, characterized in that 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. 65. The method according to any one of paragraphs. 1-64, characterized in that any said amplicon is obtained by any one or more of polymerase chain reaction (PCR), strand displacement amplification (SDA), helicase dependent amplification (HDA), recombinase polymerase amplification (RPA), loop isothermal amplification (LAMP), rolling ring 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). 66. The method according to any one of paragraphs. 1-65, characterized in that the sample is a biological sample obtained from the subject. 67. The method according to any one of paragraphs. 1-65, characterized in that the method is performed in vitro. 68. The method according to any one of paragraphs. 1-65, characterized in that the method is performed ex vivo. 69. Composition for determining the presence or absence of the first and second targets in the sample, containing: first and second closed stem-loop oligonucleotides, wherein each of the closed stem-loop oligonucleotides contains a double-stranded stem fragment formed by hybridized nucleotides and connected to a single-stranded loop fragment formed by unhybridized nucleotides, wherein: the number of hybridized nucleotides and/or the sequence of hybridized nucleotides in the stem fragment of the first and second closed stem-loop oligonucleotides are different, and each of the double-stranded stem fragments contains a fluorophore molecule attached to one strand and a quencher molecule attached to the opposite strand, moreover, the fluorophore molecules emit in the same color region of the visible spectrum, and the melting temperature (Tm) of the double-stranded stem fragment of the first closed stem-loop oligonucleotide differs from the Tm of the double-stranded stem fragment of the second closed stem-loop oligonucleotide. 70. The composition according to claim 69, characterized in that the single-stranded loop fragment of the first closed stem-loop oligonucleotide differs from the sequence of the single-stranded loop fragment of the second closed stem-loop oligonucleotide. 71. The composition according to claim 69 or 70, additionally containing: a first MNKzyme containing substrate chains capable of hybridizing to a closed single-stranded loop fragment of a first closed stem-loop oligonucleotide; and a second MNKzyme containing substrate chains capable of hybridizing to a single-stranded loop fragment of a second closed stem-loop oligonucleotide. 72. Composition according to claim 71, characterized in that: substrate chains of the first MNCzyme hybridize with a single-stranded loop fragment of the first closed stem-loop oligonucleotide; and the substrate chains of the second MNCzyme hybridize with a single-stranded loop fragment of the second closed stem-loop oligonucleotide. 73. Composition according to claim 72, characterized in that: the first and/or second MNKzymes contain an aptamer sequence associated with the analyzed target compound, protein, compound or molecule, and sensor strands hybridized to the oligonucleotide sequence; and the first MNKzyme is designed to detect a target that is different from the target of the second MNKzyme. 74. Composition according to claim 72, characterized in that: the first and/or second MNKzymes contain sensory strands that hybridize to the target sequence, and the first MNKzyme is designed to detect a target that is different from the target of the second MNKzyme. 75. The composition according to any one of paragraphs. 69-74, additionally containing: a first oligonucleotide sequence hybridizing to said single-stranded loop fragment of a first closed stem-loop oligonucleotide that differs from the sequence of a single-stranded loop fragment of a second closed stem-loop oligonucleotide, thereby forming a double-stranded sequence comprising said first oligonucleotide; a second oligonucleotide sequence hybridizing to said single-stranded loop fragment of a second closed stem-loop oligonucleotide different from the sequence of the single-stranded loop fragment of the first closed stem-loop oligonucleotide, thereby forming a double-stranded sequence comprising said second oligonucleotide; a restriction endonuclease associated with a double-stranded sequence comprising said first oligonucleotide and capable of cleaving it; and/or a restriction endonuclease associated with a double-stranded sequence comprising said second oligonucleotide and capable of cleaving it. 76. The composition according to any one of paragraphs. 69-75, additionally containing: a first double-stranded sequence comprising a first target oligonucleotide sequence hybridizing to said single-stranded loop fragment of the first closed stem-loop oligonucleotide different from the sequence of the single-stranded loop fragment of the second closed stem-loop oligonucleotide, thereby forming the first double-stranded sequence, containing the specified first oligonucleotide target; a second double-stranded sequence comprising a second target oligonucleotide sequence hybridizing to said single-stranded loop fragment of the second closed stem-loop oligonucleotide different from the sequence of the single-stranded loop fragment of the first closed stem-loop oligonucleotide, thereby forming a double-stranded sequence containing said second target oligonucleotide; a first primer oligonucleotide hybridizing to the first target oligonucleotide upstream of said first double-stranded sequence, and a first enzyme having exonuclease activity associated with a stem-loop loop of the first closed stem-loop oligonucleotide at the end of the first primer-oligonucleotide, or next to him; and/or a second primer oligonucleotide hybridizing to a second target oligonucleotide upstream of said second double stranded sequence, and a second enzyme having exonuclease activity associated with a loop fragment of the second closed stem-loop oligonucleotide at the end of the second primer oligonucleotide, or next to him. 77. Composition according to any one of paragraphs. 69-76, additionally containing: a first double-stranded sequence comprising a first target oligonucleotide sequence hybridizing to said single-stranded loop fragment of the first closed stem-loop oligonucleotide different from the sequence of the single-stranded loop fragment of the second closed stem-loop oligonucleotide, thereby forming the first double-stranded sequence, containing the specified first oligonucleotide target; a second double-stranded sequence comprising a second target oligonucleotide sequence hybridizing to said single-stranded loop fragment of the second closed stem-loop oligonucleotide different from the sequence of the single-stranded loop fragment of the first closed stem-loop oligonucleotide, thereby forming a second double-stranded sequence, containing the specified second oligonucleotide target; wherein the first enzyme having exonuclease activity is associated with the first double stranded sequence and the second enzyme having exonuclease activity is associated with the second double stranded sequence. 78. Composition according to any one of paragraphs. 69-77, additionally containing: the first DNAzyme and/or the first ribozyme hybridizing to a loop fragment of the first closed stem-loop oligonucleotide that differs from the sequence of the single-stranded loop fragment of the second closed stem-loop oligonucleotide, a second DNAzyme and/or a second ribozyme hybridizing to a loop fragment of the second closed stem-loop oligonucleotide that differs from the sequence of the single-stranded loop fragment of the first closed stem-loop oligonucleotide, cofactors for each said DNAzyme capable of conferring catalytic activity on each said DNAzyme and thus inducing the cleavage activity of any said DNAzyme hybridized to a loop fragment, cofactors for each said ribozyme capable of conferring catalytic activity on each said ribozyme and thus inducing the cleavage activity of any said loop hybridized ribozyme. 79. The composition according to any one of paragraphs. 69-78, additionally containing: third closed stem-loop oligonucleotides, wherein said third closed stem-loop oligonucleotides comprise a double-stranded stem fragment of hybridized nucleotides connected to a single-stranded loop fragment of unhybridized nucleotides, wherein: the number of hybridized nucleotides and/or the sequence of hybridized nucleotides in the stem fragment of the third closed stem-loop oligonucleotides differs from the number and sequence of hybridized nucleotides in the stem fragments of the first and second closed stem-loop oligonucleotides, and the double-stranded stem fragment of the third closed stem-loop oligonucleotides contains a fluorophore molecule connected to one strand and a quencher molecule connected to the opposite strand, and the fluorophore molecule of the third closed stem-loop oligonucleotide emits in a different region of the visible spectrum compared to with fluorophore molecules of the first and second closed stem-loop oligonucleotides. 80. The composition according to any one of paragraphs. 69-78, additionally containing: third closed stem-loop oligonucleotides, wherein said third closed stem-loop oligonucleotides comprise a double-stranded stem fragment of hybridized nucleotides connected to a single-stranded loop fragment of unhybridized nucleotides, wherein: the number of hybridized nucleotides and/or the sequence of hybridized nucleotides in the stem fragment of the third closed stem-loop oligonucleotides differs from the number and sequence of hybridized nucleotides in the stem fragments of the first and second closed stem-loop oligonucleotides, and the double-stranded stem fragment of the third closed stem-loop oligonucleotides contains a fluorophore molecule connected to one strand and a quencher molecule connected to the opposite strand, and the fluorophore molecule of the third closed stem-loop oligonucleotide emits in the same region of the visible spectrum along compared with the fluorophore molecules of the first and second closed stem-loop oligonucleotides. 81. The composition according to any one of paragraphs. 69-78, additionally containing: third closed stem-loop oligonucleotides, wherein said third closed stem-loop oligonucleotides comprise a double-stranded stem fragment of hybridized nucleotides connected to a single-stranded loop fragment of unhybridized nucleotides, wherein: the number of hybridized nucleotides and/or the sequence of hybridized nucleotides in the stem fragment of the third closed stem-loop oligonucleotides matches the number and sequence of hybridized nucleotides in the stem fragments of the first or second closed stem-loop oligonucleotides, and the double-stranded stem fragment of the third closed stem-loop oligonucleotides contains a fluorophore molecule connected to one strand and a quencher molecule connected to the opposite strand, and the fluorophore molecule of the third closed stem-loop oligonucleotide emits in a different region of the visible spectrum compared to with fluorophore molecules of the first and second closed stem-loop oligonucleotides.

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