Nicking-initiated circularization of emission and displacement reactions

Abstract

The present disclosure describes in detail a polymerase-free, isothermal method for detecting nucleotide sequences. The method uses a target-specific hairpin oligonucleotide that also specifically interacts with a probe labeled with a fluorescent dye and a quenching group, amplifying detection of the target polynucleotide through repeated cycles of endonuclease-mediated probe cleavage to drive dissociation of the fluorophore-labeled probe fragment from the hairpin-probe complex, thereby separating the fluorescent label from the quencher.

Description

Technical Field

[0001] The present invention relates to isothermal, polymerase-free methods for detecting nucleic acid sequences. Background Technology

[0002] The following describes in detail a novel method that significantly improves the speed and sensitivity of existing target nucleic acid detection methods. This method can be implemented at room temperature. Unlike RT-PCR, this method eliminates the need for expensive enzymes or cryogenic nucleotides, as well as extensive laboratory equipment for temperature cycling / imaging, thus significantly reducing costs. Limited refrigeration and equipment requirements make this method highly portable and accessible. Furthermore, the reagents used in this method can be lyophilized, further enhancing the accessibility of the technology.

[0003] The increased speed and sensitivity are achieved through the unique use of hairpin loops as hydrolysis sites, coordinating events that indicate the presence of a target and drive signal amplification after specific detection of the target sequence. The mechanism involves linking the restriction site to the target, generating a signal via the target foot or tail hairpin. This results in multiplex detection capability, partly through the use of a single target-specific hairpin, a loop sequence-specific nicking endonuclease, and a nicking endonuclease functional reaction temperature. Notably, the detection limit is at the single-molecule level, enabling single-nucleotide allele differentiation. The significant increase in fluorescence signal is mediated by the substitution of unconnected linear or exponential repeat strands on the hairpin loop and the rapid fluorescence release of the nicking endonuclease target sequence.

[0004] The method described below allows for single-molecule detection of target nucleic acids (with or without target extraction, purification, or washing) with sensitivity as low as 5 copies per reaction. This is achieved through fluorescent dye emission and nucleic acid strand displacement initiated by a nick endonuclease. This technique eliminates the need for complex equipment and reaction setups, multi-enzyme interactions, and temperature-controlled cycling. The entire reaction can be completed within 20 minutes at room temperature.

[0005] Another advantage of the methods described below is that they do not require the increased reaction temperature as qPCR, LAMP, or EXPAR methods. Furthermore, the methods described below offer higher accuracy compared to PCR-based detection methods because, unlike PCR-based methods, there is no risk of introducing amplicon contamination into the reaction or future reactions.

[0006] Other advantages of this method include: (i) reduced labor and costs due to significantly shorter sample processing and reaction times; (ii) more than three times the sample processing volume; (iii) no need for reverse transcription; and (iv) optional DNA or RNA extraction, as NICER is a method that allows for direct analysis from specimen collection. These advantages make the NICER method suitable for field applications, such as in developing countries or remote areas. Summary of the Invention

[0007] The invention described herein relates to a specific, polymerase-free, isothermal method for amplifying target nucleic acid sequences for detection.

[0008] In one aspect, the present invention provides a method for detecting one or more single-stranded target polynucleotides (target polynucleotides) in a sample, wherein each target polynucleotide comprises a first target nucleotide sequence (A sequence) and a second target nucleotide sequence (B sequence) side-joined to the first target nucleotide sequence, the method comprising the following steps: (A) Contacting the one or more target polynucleotides with a plurality of hairpin oligonucleotides, wherein each hairpin oligonucleotide comprises: (i) A hairpin structure comprising a double-stranded stem region and a single-stranded loop region (hairpin C sequence), wherein the double-stranded stem region contains a nucleotide sequence complementary to the B sequence of the target polynucleotide (hairpin B' sequence), which forms a double strand with the complementary sequence (hairpin B sequence); and (ii) A single-stranded nucleotide sequence (hairpin A' sequence) located at the 5' or 3' end of the double-stranded stem region, wherein the single-stranded nucleotide sequence comprises a nucleotide sequence complementary to the A sequence of the target polynucleotide; (B) The hairpin A' and hairpin B' sequences in one or more hairpin oligonucleotides are hybridized with the A and B sequences of one or more target polynucleotides, respectively, to form a complex of hairpin oligonucleotide and target polynucleotide, wherein the hybridization of the hairpin B' sequence with the target polynucleotide B sequence opens the stem-loop structure of the hairpin, thereby exposing a single-stranded sequence including the hairpin C sequence and the hairpin B sequence; (C) Hybridizing one or more single-stranded oligonucleotide probes (one or more probes) from a plurality of probes to the exposed single-stranded C sequence of the hairpin structure to form one or more double-stranded hybridization probe complexes, each probe comprising a nucleic acid sequence (C' sequence) complementary to the hairpin C sequence and a nucleotide sequence at a nick endonuclease (NE) site, wherein each probe is linked to: (i) a quenchable fluorescent group attached to the 3' end, internal location, or 5' end of the probe; and (ii) A quenching molecule (quencher) attached to the 3' end, internal nucleotide position or 5' end of the probe, wherein the NE site is located between the fluorescent and quenching groups; (D) Under reaction conditions that allow the one or more NEs to nick the double-stranded hybridization probe complex at one or more respective NE sites, the one or more double-stranded hybridization probe complexes are contacted with the one or more NEs, wherein the nicking reaction produces two probe fragments, each fragment dissociating from the hairpin C sequence, thereby releasing the quenchable fluorophore from the quencher and allowing: (i) The fluorescent group emits a fluorescent signal; and (ii) The hairpin C sequence hybridizes with another probe among the plurality of probes; and (E) Detect the fluorescence signal of one or more released probe fragments, wherein the fluorescence signal is correlated with the detection of target polynucleotides.

[0009] In one embodiment, the method further includes repeating steps (C) and (D) until the desired signal amplification level is reached or until the reactive components are exhausted.

[0010] In another aspect, the present invention provides a method for detecting one or more single-stranded target polynucleotides (target polynucleotides) in a sample, wherein each target polynucleotide comprises a first target nucleotide sequence (A sequence) and a second target nucleotide sequence (B sequence) side-joined to the first target nucleotide sequence, the method comprising the following steps: (A) Contacting the one or more target polynucleotides with a plurality of first hairpin oligonucleotides (HP1), wherein each HP1 comprises: (i) A hairpin structure comprising a double-stranded stem region and a single-stranded loop region (HP1 C sequence), wherein the double-stranded stem region contains a nucleotide sequence (HP1 B' sequence) complementary to the B sequence of the target polynucleotide, which forms a double strand with the complementary sequence (HP1 B sequence); and (ii) A single-stranded nucleotide sequence (HP1 A' sequence) located at the 5' or 3' end of the double-stranded stem region, wherein the single-stranded nucleotide sequence contains a nucleotide sequence complementary to the A sequence of the target polynucleotide; (B) The HP1 A' and HP1 B' sequences of one or more HP1s are hybridized with the A and B sequences of one or more target polynucleotides, respectively, to form a complex of HP1 and target polynucleotide, wherein the hybridization of the HP1 B' sequence with the B sequence of the target polynucleotide opens the stem-loop structure of HP1, thereby exposing a single-stranded sequence including the HP1 C sequence and the HP1 B sequence. (C) The complex of the HP1 with the target polynucleotide is contacted with a plurality of second hairpin oligonucleotides (HP2), wherein each HP2 comprises: (i) A hairpin structure comprising a double-stranded stem region and a single-stranded loop region, wherein: (a) The double-stranded stem region contains a nucleotide sequence complementary to the HP1 C sequence (HP2 C' sequence), which forms a double strand with the complementary sequence (HP2 C sequence); and (b) The single-stranded circular region contains a nucleotide sequence (HP2 B sequence) complementary to the HP1 B' sequence, the HP2 B sequence being flanked by a nucleotide sequence (HP2 A sequence) complementary to at least one continuous portion of the HP1 A' sequence. S sequence); (ii) Single-stranded nucleotide sequence (HP2 B') S A single-stranded nucleotide sequence located at the 5' or 3' end of the double-stranded stem region, wherein the single-stranded nucleotide sequence comprises a nucleotide sequence complementary to at least one continuous portion of the HP1 B sequence; (D) HP2 B' of one or more HP2 S HP2 C', HP2 B, and HP2 A S The sequences hybridize with the HP1 B, HP1 C, HP1 B' and HP1 A' sequences of the HP1-target polynucleotide complex, respectively, to form HP2 and HP1-target polynucleotide complexes, wherein the hybridization of HP2 with HP1 opens the stem-loop structure of HP2, thereby exposing a single-stranded sequence including the HP2 C sequence. (E) Hybridizing one or more single-stranded oligonucleotide probes (one or more probes) from a plurality of probes with the exposed HP1 C sequence and the exposed single-stranded HP2 sequence of the HP2 and HP1 complex with the target polynucleotide to form one or more double-stranded hybridization probe complexes, each probe comprising a nucleic acid sequence (C' sequence) complementary to the HP1 C sequence and a nucleotide sequence at a nick endonuclease (NE) site, wherein each probe is linked to: (i) a quenchable fluorescent group attached to the 3' end, internal location, or 5' end of the probe; and (ii) A quenching molecule (quencher) attached to the 3' end, internal nucleotide position or 5' end of the probe, wherein the NE site is located between the fluorescent and quenching groups; (F) Under reaction conditions that allow the one or more NEs to nick the double-stranded hybridization probe complex at one or more respective NE sites, the one or more double-stranded hybridization probe complexes are contacted with the one or more NEs, wherein the nicking reaction produces two probe fragments, each fragment dissociating from the hairpin C sequence, thereby releasing the quenchable fluorophore from the quencher and allowing: (i) The fluorescent group emits a fluorescent signal; and (ii) The exposed HP1 and exposed single-stranded HP2 C sequences hybridize with another probe among the plurality of probes; and (G) Detect the fluorescence signal of one or more released probe fragments, wherein the fluorescence signal is correlated with the detection of target polynucleotides.

[0011] In some embodiments, the HP2 C' sequence may contain at least one thiophosphate modification.

[0012] In another aspect, the present invention provides a method for detecting one or more single-stranded target polynucleotides (target polynucleotides) in a sample, wherein each target polynucleotide comprises a first target nucleotide sequence (A sequence) and a second target nucleotide sequence (B sequence) side-joined to the first target nucleotide sequence, the method comprising the following steps: (A) Contacting the one or more target polynucleotides with a plurality of first hairpin oligonucleotides (HP1), wherein each HP1 comprises: (i) A hairpin structure comprising a double-stranded stem region and a single-stranded loop region (HP1 C sequence), wherein the double-stranded stem region contains a nucleotide sequence (HP1 B' sequence) complementary to the B sequence of the target polynucleotide, which forms a double strand with the complementary sequence (HP1 B sequence), wherein the HP1 B sequence contains a nucleotide sequence at a first nick endonuclease (NE1) site; and (ii) A single-stranded nucleotide sequence (HP1 A' sequence) located at the 5' end of the double-stranded stem region, wherein the single-stranded nucleotide sequence contains a nucleotide sequence complementary to the A sequence of the target polynucleotide; (B) The HP1 A' and HP1 B' sequences of one or more HP1s are hybridized with the A and B sequences of one or more target polynucleotides, respectively, to form a complex of HP1 and target polynucleotide, wherein the hybridization of the HP1 B' sequence with the B sequence of the target polynucleotide opens the stem-loop structure of HP1, thereby exposing a single-stranded sequence including the HP1 C sequence and the HP1 B sequence. (C) The complex of the HP1 with the target polynucleotide is contacted with a plurality of second hairpin oligonucleotides (HP2), wherein each HP2 comprises: (i) A hairpin structure comprising a double-stranded stem region and a single-stranded loop region (HP2 A sequence), wherein the double-stranded stem region comprises a nucleotide sequence complementary to the HP1 B sequence (HP2 B' sequence), which forms a double strand with the complementary sequence (HP2 B sequence); and (ii) A single-stranded nucleotide sequence (HP2 C' sequence) located at the 3' end of the double-stranded stem region, wherein the single-stranded nucleotide sequence comprises a nucleotide sequence complementary to the HP1 C sequence and a nucleotide sequence at the second NE (NE2) site; (D) The HP2 C' and HP2 B' sequences of one or more HP2s are hybridized with the exposed single-stranded HP1 C and HP1 B sequences of the HP1-target polynucleotide complex, respectively, to form HP2 and HP1-target polynucleotide complexes, wherein the hybridization of HP2 with HP1 opens the stem-loop structure of HP2, thereby exposing single-stranded sequences including HP2 A and HP2 B sequences; (E) Hybridizing one or more single-stranded oligonucleotide probes (one or more probes) from a plurality of probes with an exposed HP1 C sequence to form one or more double-stranded hybridization probe complexes, each probe comprising a nucleic acid sequence (C' sequence) complementary to the HP1 C sequence and a nucleotide sequence at the NE2 site, wherein each probe is linked with: (i) a quenchable fluorescent group attached to the 3' end, internal location, or 5' end of the probe; and (ii) A quenching molecule (quencher) attached to the 3' end, internal nucleotide position or 5' end of the probe, wherein the NE2 site is located between the fluorescent and quenching groups; (F) Contact the one or more double-stranded hybridization probe complexes with the one or more NE2s under reaction conditions that allow the one or more NE2s to perform the following operations: (i) The double-stranded hybridization probe complex is nicked at one or more respective NE2 sites, wherein the nicking reaction produces two probe fragments, each fragment dissociating from the hairpin C sequence, thereby releasing the quenchable fluorophore from the quencher and allowing: (a) The fluorescent group emits a fluorescent signal; and (b) The exposed HP1 and exposed single-stranded HP2 C sequences hybridize with another probe among the plurality of probes; and; (ii) cleaving the NE2 site of the HP2 C' sequence of the complex of the HP2 and HP1 with the target polynucleotide, thereby allowing: (a) Dissociation of the short 3' HP2 C' segment; and (b) A long HP2 fragment comprising the 5' HP2 C' sequence and HP2 B', A and B sequences is replaced by another HP2; (G) Under reaction conditions that allow the one or more NE1s to cleave the NE1 site of the HP1 B sequence of the HP2 and HP1 complex with the target polynucleotide, the HP2 and HP1 complex with the target polynucleotide is contacted with the one or more NE1s, thereby allowing the cleaved HP1 B fragment to dissociate. (H) Detect the fluorescence signal of one or more released probe fragments, wherein the fluorescence signal is correlated with the detection of target polynucleotides.

[0013] In one implementation, the method further includes the following steps: (I) The long HP2 fragment comprising the 5' HP2 C' sequence and HP2 B', A, and B sequences is contacted with another HP1 from a plurality of HP1s, thereby hybridizing the HP1 A' and HP1 B' sequences of the one or more HP1s with the HP2 A and HP2 B sequences, respectively, to form an HP1-HP2 complex, wherein the hybridization of the HP1 B' sequence with the HP2 B sequence opens the stem-loop structure of the HP1, thereby exposing the single-stranded sequences comprising the HP1 C and HP1 B sequences; and (J) Repeat steps (D)-(H) until the desired signal amplification level is reached or until the reactants are exhausted.

[0014] In another aspect, the present invention provides a method for detecting one or more single-stranded target polynucleotides (target polynucleotides) in a sample, wherein each target polynucleotide comprises a first target nucleotide sequence (A sequence) and a second target nucleotide sequence (B sequence) side-joined to the first target nucleotide sequence, the method comprising the following steps: (A) Contacting the one or more target polynucleotides with a plurality of first hairpin oligonucleotides (HP1), wherein each HP1 comprises: (i) A hairpin structure comprising a double-stranded stem region and a single-stranded loop region (HP1 C sequence), wherein the double-stranded stem region contains a nucleotide sequence (HP1 B' sequence) complementary to the B sequence of the target polynucleotide, which forms a double strand with the complementary sequence (HP1 B sequence); and (ii) A single-stranded nucleotide sequence (HP1 A' sequence) located at the 5' or 3' end of the double-stranded stem region, wherein the single-stranded nucleotide sequence contains a nucleotide sequence complementary to the A sequence of the target polynucleotide; (B) The HP1 A' and HP1 B' sequences of one or more HP1s are hybridized with the A and B sequences of one or more target polynucleotides, respectively, to form a complex of HP1 and target polynucleotide, wherein the hybridization of the HP1 B' sequence with the B sequence of the target polynucleotide opens the stem-loop structure of HP1, thereby exposing a single-stranded sequence including the HP1 C sequence and the HP1 B sequence. (C) The complex of the HP1 with the target polynucleotide is contacted with a plurality of second hairpin oligonucleotides (HP2), wherein each HP2 comprises: (i) A hairpin structure comprising a double-stranded stem region and a single-stranded loop region (HP2 A sequence), wherein the double-stranded stem region contains a nucleotide sequence complementary to the HP1 B sequence (HP2 B sequence), which forms a double strand with the complementary sequence (HP2 B' sequence); (ii) A single-stranded nucleotide sequence (HP2 C' sequence) located at the 5' end of the double-stranded stem region, wherein the single-stranded nucleotide sequence comprises a nucleotide sequence complementary to the HP1 C sequence and a nucleotide sequence at the first nick endonuclease (NE1) site; and (iii) The nucleotide sequence of the second NE (NE2) site, located between the HP2 B sequence and the HP2 C' sequence; (D) The HP2 C', HP2 B' and HP2 A sequences of one or more HP2 are hybridized with the exposed single-stranded HP1 C sequence, HP1 B' sequence and HP1 A' sequence of the HP1-target polynucleotide complex, respectively, so that HP1 dissociates from the target polynucleotide and forms a complex of HP2 and HP1, wherein the hybridization of HP2 and HP1 opens the stem-loop structure of HP2, thereby exposing the single-stranded sequence including the HP2 B' sequence; (E) Hybridizing one or more single-stranded oligonucleotide probes (one or more probes) from a plurality of probes with an exposed HP1 C sequence to form one or more double-stranded hybridization probe complexes, each probe comprising a nucleic acid sequence (C' sequence) complementary to the HP1 C sequence and a nucleotide sequence at the NE1 site, wherein each probe is linked with: (i) a quenchable fluorescent group attached to the 3' end, internal location, or 5' end of the probe; and (ii) A quenching molecule (quencher) attached to the 3' end, an internal nucleotide position, or the 5' end of the probe, wherein the NE1 site is located between the fluorescent and quenching groups; (F) Contact the one or more double-stranded hybridization probe complexes with the one or more NE1s under reaction conditions that allow the one or more NE1s to perform the following operations: (i) The double-stranded hybridization probe complex is nicked at one or more respective NE1 sites, wherein the nicking reaction produces two probe fragments, each fragment dissociating from the hairpin C sequence, thereby releasing the quenchable fluorophore from the quencher and allowing: (a) The fluorescent group emits a fluorescent signal; and (b) The exposed HP1 and exposed single-stranded HP2 C sequences hybridize with another probe among the plurality of probes; and; (ii) cleaving the NE1 site of the HP2 C' sequence of the HP2 and HP1 complex to allow dissociation of the short 5' HP2 C' fragment; and (G) Under reactive conditions that allow the one or more NE2s to cleave the NE2 site located between the HP2 B sequence and the HP2 C' sequence, the complex of HP2 and HP1 is contacted with the one or more NE2s, thereby allowing the 3' HP2 C' fragment of the cleavage to dissociate and form the complex of the cleavage HP2 and HP1. (H) Contact another HP2 from the plurality of HP2s with the HP1 of the complex of HP2 and HP1 of the cut, thereby hybridizing the HP2 C' sequence, HP2 B' sequence and HP2 A sequence of the one or more HP2s with the exposed single-stranded HP1C sequence, HP1 B' sequence and HP1 A' sequence, respectively, replacing the cut HP2 including the HP2 B, HP2 A and HPB' sequences; (I) Detect the fluorescence signal of one or more released probe fragments, wherein the fluorescence signal is correlated with the detection of target polynucleotides.

[0015] In some embodiments, the HP2 B' sequence of the substituted HP2 in step (H) hybridizes with the exposed single-stranded HP1 sequence formed in step (B), thereby allowing hybridization of the HP1 A' and HP1 B' sequences of another HP1 from the plurality of HP1s, wherein hybridization of the HP1 B' sequence with the HP2 B sequence of the HP2 fragment B opens the stem-loop structure of HP1, thereby exposing the single-stranded sequence including the HP1 C and HP1 B sequences. The method further includes repeating steps (D)-(H) until the desired signal amplification level is reached or until the reaction components are exhausted.

[0016] In some implementations, the methods disclosed herein are carried out under isothermal conditions.

[0017] In some implementations, the methods disclosed herein are performed at room temperature.

[0018] In another aspect, the present invention relates to a polynucleotide comprising a hairpin structure and a sequence of A', B', C, and B sequentially from 5' to 3' or from 3' to 5', wherein: The A' sequence contains a major target-specific nucleotide sequence; The B' sequence contains a minor target-specific nucleotide sequence; The C sequence contains the nucleotide sequence of the endonuclease recognition site; The B sequence comprises a nucleotide sequence complementary to the B' sequence, and optionally includes a nucleotide sequence representing an endonuclease recognition site; and The B' and B sequences hybridize to form a stem-loop structure, wherein the loop contains a C sequence.

[0019] In some embodiments of the polynucleotide hairpin, the A' sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

[0020] In some embodiments of the polynucleotide hairpin, the B' and B sequences contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

[0021] In some embodiments of the polynucleotide hairpin, the C sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

[0022] In some embodiments of the polynucleotide hairpin, the nick endonuclease recognition site is the site required for nicking using Nt.BspQI, Nt.CviPII, Nt.BstNBI, Nb.BsrDI, Nb.BtsI, Nt.AlwI, Nb.BbvCI, Nt.BbvCI, Nb.BsmI, Nb.BssSI, or Nt.BsmAI.

[0023] In another aspect, the present invention is a device comprising a hairpin structure and B's arranged sequentially from 5' to 3' or from 3' to 5'. S Sequence, C' sequence, B sequence, A S Polynucleotides of sequence and C sequence, wherein: (a) The B sequence contains the same nucleotide sequence as the secondary target-specific nucleotide sequence; (b) The B' mentioned S The sequence contains a sequence complementary to the B sequence, but with fewer nucleotides than the B sequence; (c) The C' sequence comprises a nucleotide sequence of an endonuclease recognition site and at least one modified nucleotide; (d) A S The sequence contains at least one continuous portion of the same nucleotide sequence as the primary target-specific sequence; (e) The C' and C sequences hybridize to form a stem-loop structure, wherein the loop contains the B sequence and the A sequence. S sequence; and (f) The C' sequence contains at least one modified nucleotide that prevents nick endonuclease activity at the nick endonuclease recognition site nucleotide.

[0024] In some embodiments of the polynucleotide hairpin, the A sequence comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

[0025] In some embodiments of the polynucleotide hairpin, the B sequence comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

[0026] In some embodiments of the polynucleotide hairpin, the C and C' sequences comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

[0027] In some embodiments of the polynucleotide hairpin, the modified nucleic acid in (e) is a phosphate thioester-modified nucleic acid, peptide nucleic acid (PNA), locked nucleic acid (LNA), dNTP / ribonucleoside triphosphate (rNTP) hybrid, isoguanosine (isoG); isocytidine (isoC), dUTP, rATP, rCTP, rGTP, or rUTP.

[0028] In some embodiments of the polynucleotide hairpin, the at least one modified nucleic acid is located at: 1-4 nucleotides from the 5' end, at an internal nucleotide position, or at or near the 3' end.

[0029] In another aspect, the present invention relates to a polynucleotide comprising a hairpin structure and a sequence consisting of a C' sequence, a B sequence, an A sequence, and a B' sequence from 5' to 3', wherein: (a) The A sequence contains a major target-specific nucleotide sequence; (b) The B sequence comprises a minor target-specific nucleotide sequence and a first nick endonuclease recognition site nucleotide sequence; (c) The B' sequence contains a nucleotide sequence complementary to the B sequence; (d) The B and B' sequences hybridize to form a stem-loop structure, wherein the loop contains the A sequence; and (e) The C' sequence contains the nucleotide sequence of the second nick endonuclease recognition site.

[0030] In another aspect, the present invention is a polynucleotide comprising a hairpin structure and sequences B, A, B', and C' arranged sequentially from 5' to 3', wherein: (a) The A sequence contains a major target-specific nucleotide sequence; (b) The B sequence contains a minor target-specific nucleotide sequence; (c) The B' sequence contains a nucleotide sequence complementary to the B sequence; (d) The B and B' sequences hybridize to form a stem-loop structure, wherein the loop contains the A sequence; (e) The junction of the C' and B sequences contains a nucleotide sequence representing the second nick endonuclease recognition site; and (f) The C' sequence contains the nucleotide sequence of the second nick endonuclease recognition site.

[0031] In some embodiments of the polynucleotide hairpin, the A sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

[0032] In some embodiments of the polynucleotide hairpin, the B and B' sequences contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

[0033] In some embodiments of the polynucleotide hairpin, the C' sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

[0034] In some embodiments of the polynucleotide hairpin, the nick endonuclease recognition site, or the first nick endonuclease recognition site and the second nick endonuclease recognition site, are independently the sites required for nicking Nt.BspQI, Nt.CviPII, Nt.BstNBI, Nb.BsrDI, Nb.BtsI, Nt.AlwI, Nb.BbvCI, Nt.BbvCI, Nb.BsmI, Nb.BssSI, or Nt.BsmAI.

[0035] In another aspect, the present invention is a linear single-stranded polynucleotide probe comprising a C' sequence, a dye molecule, and a quencher molecule, wherein: The C' sequence comprises, sequentially from 5' to 3' or from 3' to 5': The optional X sequence may consist of 1-8 nucleotides; Nucleotide sequence of the endonuclease recognition site; and The optional Y sequence consists of 1-8 nucleotides.

[0036] In some embodiments of the polynucleotide probe, the nicking endonuclease recognition site is the site required for nicking by Nt.BspQI, Nt.CviPII, Nt.BstNBI, Nb.BsrDI, Nb.BtsI, Nt.AlwI, Nb.BbvCI, Nt.BbvCI, Nb.BsmI, Nb.BssSI, or Nt.BsmAI.

[0037] In some embodiments of the polynucleotide probe, the probe contains an X sequence.

[0038] In some embodiments of the polynucleotide probe, the X sequence is labeled with a dye, wherein the dye is located inside the X sequence; if the X sequence is located at the 5' end of the C' sequence, it is located at or near the 5' end of the probe; or, if the X sequence is located at the 3' end of the C' sequence, it is located at or near the 3' end of the probe.

[0039] In some embodiments of the polynucleotide probe, the dye is 6-carboxyfluorescein (6-FAM) or carboxy-X-rhodamine (ROX).

[0040] In some embodiments of the polynucleotide probe, the X sequence is labeled with a quenching group, wherein the quenching group is located inside the X sequence; if the X sequence is located at the 5' end of the C' sequence, it is located at or near the 5' end of the probe; or, if the X sequence is located at the 3' end of the C' sequence, it is located at or near the 3' end of the probe.

[0041] In some embodiments of the polynucleotide probe, the quenching group is a black hole quencher (BHQ).

[0042] In some embodiments of the polynucleotide probe, the probe contains a Y sequence.

[0043] In some embodiments of the polynucleotide probe, the Y sequence is labeled with a dye, wherein the dye is located inside the Y sequence; if the Y sequence is located at the 5' end of the C' sequence, it is located at or near the 5' end of the probe; or, if the Y sequence is located at the 3' end of the C' sequence, it is located at or near the 3' end of the probe.

[0044] In some embodiments of the polynucleotide probe, the dye is 6-carboxyfluorescein (6-FAM) or carboxy-X-rhodamine.

[0045] In some embodiments of the polynucleotide probe, the Y sequence is labeled with a quenching group, wherein the quenching group is located inside the Y sequence; if the Y sequence is located at the 5' end of the C' sequence, it is located at or near the 5' end of the probe; or, if the Y sequence is located at the 3' end of the C' sequence, it is located at or near the 3' end of the probe.

[0046] In some embodiments of the polynucleotide probe, the quenching group is a black hole quencher (BHQ).

[0047] In another aspect, the present invention is a linear single-stranded polynucleotide probe comprising a C' sequence, a dye molecule, and a quencher molecule, wherein: The C' sequence comprises, sequentially from 5' to 3' or from 3' to 5': The optional X sequence may consist of 1-4 nucleotides; The first nucleotide sequence of the endonuclease recognition site; The optional Y sequence includes 1-4 nucleotides; The nucleotide sequence of the second nick endonuclease recognition site; and The optional Z sequence consists of 1-4 nucleotides.

[0048] In some embodiments of the polynucleotide probe, the nicking endonuclease recognition site is the site required for nicking by Nt.BspQI, Nt.CviPII, Nt.BstNBI, Nb.BsrDI, Nb.BtsI, Nt.AlwI, Nb.BbvCI, Nt.BbvCI, Nb.BsmI, Nb.BssSI, or Nt.BsmAI.

[0049] In some embodiments of the polynucleotide probe, the probe contains an X sequence.

[0050] In some embodiments of the polynucleotide probe, the X sequence is labeled with a dye, wherein the dye is located inside the X sequence; if the X sequence is located at the 5' end of the C' sequence, it is located at or near the 5' end of the probe; or, if the X sequence is located at the 3' end of the C' sequence, it is located at or near the 3' end of the probe.

[0051] In some embodiments of the polynucleotide probe, the dye is 6-carboxyfluorescein (6-FAM) or carboxy-X-rhodamine (ROX).

[0052] In some embodiments of the polynucleotide probe, the X sequence is labeled with a quenching group, wherein the quenching group is located inside the X sequence; if the X sequence is located at the 5' end of the C' sequence, it is located at or near the 5' end of the probe; or, if the X sequence is located at the 3' end of the C' sequence, it is located at or near the 3' end of the probe.

[0053] In some embodiments of the polynucleotide probe, the quenching group is a black hole quencher (BHQ).

[0054] In some embodiments of the polynucleotide probe, the probe contains a Y sequence.

[0055] In some embodiments of the polynucleotide probe, the Y sequence is labeled with a dye, wherein the dye is located inside the Y sequence; if the Y sequence is located at the 5' end of the C' sequence, it is located at or near the 5' end of the probe; or, if the Y sequence is located at the 3' end of the C' sequence, it is located at or near the 3' end of the probe.

[0056] In some embodiments of the polynucleotide probe, the dye is 6-carboxyfluorescein (6-FAM) or carboxy-X-rhodamine.

[0057] In some embodiments of the polynucleotide probe, the Y sequence is labeled with a quenching group, wherein the quenching group is located inside the Y sequence; if the Y sequence is located at the 5' end of the C' sequence, it is located at or near the 5' end of the probe; or, if the Y sequence is located at the 3' end of the C' sequence, it is located at or near the 3' end of the probe.

[0058] In some embodiments of the polynucleotide probe, the quenching group is a black hole quencher (BHQ). Attached Figure Description

[0059] Figure 1 The 5' and 3' versions of the target-specific hairpin used in the linear NICER method (Method 0.0) are depicted. The A' sequence (A') is the primary target site-specific sequence (A sequence), typically 6–24 nucleotides in length. The B' sequence is the secondary target site-specific sequence (B sequence), located at the 3' or 5' end of the primary target site A. In a closed hairpin, B' hybridizes with its complementary sequence B to form a stem-loop structure, where the loop includes the C sequence (C). C contains cleavage sites partially located in the stem of the hairpin or entirely within the loop. X and Y are optional linker sequences located between B' and C or between B and C, typically 1–10 nucleotides in length.

[0060] Figure 2 The 5' and 3' versions of the probe signal substrate nucleotide sequence used in the linear NICER method are depicted. B' is optional and can be fully, partially, or absent. When B' is partially or fully present, B' hybridizes with the B sequence of the hairpin after the A' and B' of the hairpin hybridize with the A and B target sites, and then hybridizes with the B sequence of the hairpin when the hairpin opens to expose B. X' and Y' are optional sequences complementary to the X and Y sequences of the hairpin, and they are typically 1 to 10 nucleotides in length. C' contains the cleavage endonuclease catalytic site, indicated by the tone mark (^). Z is an optional sequence that, if present, side-joins B', or, if the probe does not include B' or includes a portion of the B' sequence, includes nucleotides that are not complementary to the B sequence (negative bases). Any one or more of B', C', X', Y', or Z may be labeled with a fluorescent dye and / or quencher, the fluorescent dye and / or quencher label being located at or near the 5' end, 3' end, or inside the cell, and may optionally include modified nucleotides, such as locked nucleic acids (LNAs).

[0061] Figure 3Hybridization of the 5' tail target-specific hairpin of the linear NICER probe was depicted. Hybridization of hairpin A' with RNA target A led to RNA B strand invasion and migration to hairpin B, thereby causing the hairpin to open and expose hairpin C and B.

[0062] Figure 4 This depicts the hybridization of probe 1 with the exposed C and B sequences of the hairpin after hybridization of the hairpin's A' and B' sequences with probe's A and B sequences. The probe's C' sequence contains a recognition site for a specific nicking endonuclease that nicks the probe after hybridization with the exposed A' and B' sequences of the hairpin. An asterisk represents a fluorophore, and Q represents a quencher.

[0063] Figure 5 The study describes how, after the hybridization probe is nicked, the release of the C' probe fragment in the linear NICER method causes the probe's fluorophore to dissociate from the nicked portion of the hairpin, thereby separating the fluorophore from the quencher and generating fluorescence.

[0064] Figure 6 Depicting in such Figure 5 Following the nicking reaction, the intact fluorophore-labeled probe hybridizes with the exposed single-stranded portion of the hairpin C. Hybridization of the new probe induces strand invasion and displaces the previously nicked probe from the composite hairpin-target complex.

[0065] Figure 7 The increase in fluorescence signal was depicted as the nicking and chain displacement events repeated until the reaction terminated.

[0066] Figure 8A This is a signal generation graph showing the specific detection of SARS-CoV-2 N1 (nucleocapsid) RNA using the linear probe NICER method. Samples were diluted to concentrations of 40, 20, 18, 16, 14, 12, 10, 5, and 1 viral genome / genome per microliter.

[0067] Figure 8B Is with Figure 8A The corresponding signal generation diagram uses the reaction without target RNA. Three negative controls were used to replicate the experiment.

[0068] Figure 8C The graph shows the limit of detection (LoD) of 40cp / μL SARS-CoV-2 N1 (nucleocapsid) target DNA / μL in a triple replicate test.

[0069] Figure 8D The LoD triplet test plot shows 20cp / μL SARS-CoV-2 N1 (nucleocapsid) target DNA / μL.

[0070] Figure 8EThe LoD triplet test plot shows 18cp / μL SARS-CoV-2 N1 (nucleocapsid) target DNA / μL.

[0071] Figure 8F The LoD triplet test plot shows 16cp / μL SARS-CoV-2 N1 (nucleocapsid) target DNA / μL.

[0072] Figure 8G The LoD triplet test plot shows 14cp / μL SARS-CoV-2 N1 (nucleocapsid) target DNA / μL.

[0073] Figure 8H The LoD triplet test plot shows 12cp / μL SARS-CoV-2 N1 (nucleocapsid) target DNA / μL.

[0074] Figure 8I The LoD triplet test plot shows 10cp / μL SARS-CoV-2 N1 (nucleocapsid) target DNA / μL.

[0075] Figure 8J The LoD triplet test plot shows 5cp / μL SARS-CoV-2 N1 (nucleocapsid) target DNA / μL.

[0076] Figure 8K The LoD triplet test plot shows 1cp / μL SARS-CoV-2 N1 (nucleocapsid) target DNA / μL.

[0077] Figure 9A The signal generation diagram for the control experiment without the target nucleic acid is shown to determine the appropriate hairpin concentration to minimize the possibility of background noise caused by oligonucleotide impurities. The reaction conditions were 25°C and the hairpin reaction concentration was 50 nM.

[0078] Figure 9B The signal generation diagram for the control experiment without the target nucleic acid is shown to determine the appropriate hairpin concentration to minimize the possibility of background noise caused by oligonucleotide impurities. The reaction conditions were 25°C and the hairpin reaction concentration was 25 nM.

[0079] Figure 9C The signal generation diagram for the control experiment without the target nucleic acid is shown to determine the appropriate hairpin concentration to minimize the possibility of background noise caused by oligonucleotide impurities. The reaction conditions were 25°C and the hairpin reaction concentration was 10 nM.

[0080] Figure 9DThe signal generation diagram for the control experiment without the target nucleic acid is shown to determine the appropriate hairpin concentration to minimize the possibility of background noise caused by oligonucleotide impurities. The reaction conditions were 25°C and the hairpin reaction concentration was 5 nM.

[0081] Figure 10A This is a signal generation diagram illustrating the failed fluorescence detection of SARS-CoV-2 RNA using a NICER hairpin specific to the SARS-CoV-2 B.1.1.7 - N501Y variant. The signal pattern is atypical, and signal generation collapses over time, forming a dome-shaped pattern.

[0082] Figure 10B Is with Figure 10A The corresponding signal generation diagram for the negative control results, using a reaction without target RNA.

[0083] Figure 10C yes Figure 8A , Figure 8B , Figure 10A and Figure 10B The merged signal generation diagram of the data.

[0084] Figure 11 A variant of the NICER method (Method 2) is described, which also includes linear probes, such as... Figure 1-1 As described in 0. Both linear probes and hairpin probes can hybridize with an open hairpin to induce displacement. This method exponentially amplifies the signal.

[0085] Figure 12 The accelerated signal amplification is described, which is a result of using probes containing target sequences to increase the number of hybridization and nicking events. Figure 15 A universal probe structure is shown, in which the nick nuclease site (NS) is flanked by X and Y domains that can be used to distinguish the probe in multiple versions of NICER. The X and Y domains typically each contain 0–8 nucleotides. Universal probes are typically modified by attaching dyes and / or quenchers to the 5' end, internal position, and 3' end.

[0086] Figure 13 The structure of the dual-enzyme probe was described, wherein C' consists of X' (0-4 nucleotides); NS1 includes the cleavage site of the first endonuclease; Y' includes 0-4 nucleotides; NS2 includes the cleavage site of the second endonuclease; and Z' includes 0-4 nucleotides. X', Y', or Z' may consist of more than 5 nucleotides. The probe described herein may contain a dye, quencher, or a combination thereof located at the 5' end, internal position, or 3' end.

[0087] Figure 14The structure of the dual-enzyme probe was described, wherein C' consists of X' (0-4 nucleotides); NS1 includes the cleavage site of the first endonuclease; Y' includes 0-4 nucleotides; NS2 includes the cleavage site of the second endonuclease; and Z' includes 0-4 nucleotides. X', Y', or Z' may consist of more than 5 nucleotides. The probe described herein may contain a dye, quencher, or a combination thereof located at the 5' end, internal position, or 3' end.

[0088] Figure 15 A NICER version using a universal probe (NICER method 0.5) was described. Target-specific A' and B' hairpin sequences hybridize with target sequences A and B, leading to stem opening and exposing C and B, thus allowing the probe's C' to hybridize with the hairpin's C. After a nick reaction and separation of the labeled C' probe fragment, a new universal probe hybridizes with the exposed portion of the hairpin's C, and the cycle is repeated.

[0089] Figure 16 A double-hairpin, accelerated version of NICER (NICER method 1) is depicted. Hairpin 1 (HP1) contains sequences A, B, B', and C, where B and B' are laterally attached to C and cross-link to form a stem structure. Hairpin 2 (HP2) contains a shortened B' sequence (B' S ), C, C' and B sequences, and the shortened A sequence (A S C and C' are connected to B and A respectively. S The probes hybridize to form a stem structure. HP1's A' hybridizes with the target template sequence's A, undergoing strand displacement via a hairpin, while HP1's B' hybridizes with the target template sequence's B. HP1's C and B become single-stranded, allowing the universal probe's C' to hybridize with HP1's exposed C sequence. After the probe fragment dissociates from the nicking reaction, a new, unnicked probe hybridizes with the exposed HP1 C sequence. HP2's B'... S The sequence hybridizes with HP1 B, resulting in strand substitution between HP1 and HP2. HP1 dissociates from the template sequence, and HP2 hybridizes with HP1. Subsequently, HP2 hybridizes with the C, B', and A' strands of HP1. The original template can be used to hybridize with new HP1 sequences, and the steps are repeated cyclically.

[0090] Figure 17An exponential version of NICER (“Method 4.2”) is described, comprising a double hairpin, a universal probe, and two endonuclease sites / enzymes. Method 4.2 NICER comprises a first hairpin (HP1) with a 5' protrusion and a second hairpin (HP2) with a 3' protrusion. In this method, (i) the A' of hairpin 1 (HP1) hybridizes with the target template sequence A; (ii) HP1 undergoes strand substitution; (iii) HP1 B' hybridizes with the target sequence B; (iv) HP1 C and B become single-stranded; (v) probe C' hybridizes with the exposed C sequence of HP1; (vi) the first nick endonuclease nicks the probe at the first recognition site, producing two fragments dissociated from HP1; and (vii) the new unnicked probe hybridizes with the exposed C sequence of HP1, (v)-(vii) repeated and cycled; (viii) the C' of hairpin 2 (HP2) hybridizes with the exposed C sequence of HP1; (ix) HP2 undergoes strand substitution, H... P2's B' hybridizes with HP1's B; (x) the exposed A and B sequences of HP2 serve as target sequences for unhybridized HP1; (xi) the first endonuclease cleaves at the C' recognition site of HP2, and a new uncleaved HP2 replaces the cleaved HP2; (xii) the second endonuclease cleaves at the second cleavage endonuclease recognition site in B', allowing the dissociation of the short double-stranded sequence generated by the C' cleavage of HP1 and the B' cleavage of HP2, thereby generating a free target template sequence on the resulting large fragment of HP2.

[0091] Figure 18An optional iteration of the exponential NICER method (NICER method 4.1) is described. This method uses a first hairpin and a second hairpin, both with 5' protrusions. In the reaction of method 4.1, (i) the A' of HP1 hybridizes with the A of the target template sequence; (ii) HP1 undergoes strand substitution, and the B' of HP1 hybridizes with the B of the target template sequence; (iii) the C and B of HP1 become single-stranded; (iv) the C' probe can bind to the exposed C portion of HP1; (v) the first nick endonuclease nicks the recognition site on the probe, producing two fragments; (vi) these fragments dissociate from HP1; (vii) a new unnicked probe binds to the exposed C portion of HP1; (iv)-(vi) are repeated and cycled; (viii) the C' of HP2 hybridizes with the C of HP1; (ix) HP1 and HP2 undergo strand substitution, and HP1 dissociates from the template sequence. HP2 hybridizes with HP1, and the B and A of HP2 hybridize with the B' and A' of HP1; (x) the original template sequence present on HP2 can be used for new HP1 hybridization; (xi) the C' of HP2 can be cleaved by the first and second endonucleases to produce small fragments, each fragment having a similar Tm below 25°C, thus dissociating from HP1; (xii) a new, uncleaved HP2 can invade and hybridize with HP1, replacing the cleaved HP2; and (xiii) the replaced HP2 large fragment can form a hairpin, or the B' of HP2 can hybridize with the exposed B of HP1, keeping the cleaved HP2 open and providing a new target template sequence for the invasion of unhybridized HP1.

[0092] Figure 19 This is a signal generation diagram, showing the signal generation between a probe with an internal quencher and a probe with an internal dye.

[0093] Figure 20 This demonstrates the signal generation between Method 0 (linear method, hairpin-specific probe) and Method 0.5 (linear, general probe).

[0094] Figure 21 This is a signal generation graph showing the results of testing NICER method 0.5 with different template concentrations using lyophilized NICER premix.

[0095] Figure 22 The top image shows the signal generation plot for Method 0.5, illustrating the differences in signal generation relative to different universal probe concentrations. The bottom image shows the signal generation plot for Method 0.5, illustrating the differences in signal generation relative to different hairpin concentrations.

[0096] Figure 23Multiple NICER signal amplification data for two different targets are shown, including mismatched hairpin and probe combinations, to demonstrate specificity. (1) Templates matching FAM hairpin and probe exist; (2) Templates matching ROX hairpin and probe exist; (3) Templates matching FAM hairpin and probe exist; (4) Templates matching ROX hairpin and probe exist; (5) Templates matching FAM hairpin and probe exist; (6) Templates not matching ROX hairpin and FAM probe exist. (7) Templates matching ROX hairpin and probe exist; (8) Templates not matching FAM hairpin and ROX probe exist.

[0097] Figure 24 Data obtained using the NICER 0.5 method are shown for comparison, with reaction mixtures containing varying amounts of 6000MW PEG. Experimental conditions included CFX96, 1s x 250 cycles (excluding imaging), and a premix with all components added to the template. Bar graphs show how template and PEG concentrations affect signal amplification.

[0098] Figure 25 The data shown are from comparisons to the NICER 0.5 method, where the reaction mixture contained 6000 MW or 8000 MW of PEG or no PEG. Bar graphs show how template concentration and PEG concentration affect signal amplification.

[0099] Figure 26 This is a signal generation diagram, MW in CFX96, 25. C, 1s cycle x 250 cycles (excluding imaging), signal generation comparison between Method 0.5 and Method 1 using 50nM template, 50nM hairpin, and 50nM probe.

[0100] Figure 27 This is a signal generation diagram showing a comparison of the background signal detected when using a probe with an end quencher and dye or a probe with an internal quencher and end dye.

[0101] Figure 28 The image above shows the signal generation plots, comparing the background signals generated by the modified hairpin 2 in Method 1 NICER (top). The background signal (gray) of the template-free sample is indistinguishable from that of the template-containing sample (black). The image below shows the signal generation plots of Method 1 NICER using the modified hairpin 2 to protect the notch sites in its C' domain. There is a clear difference between the background signal (gray) of the template-free sample and that of the template-containing sample (black). Detailed Implementation

[0102] This invention provides details of a polymerase-free isothermal method for amplifying the number of signal events generated after repeated cycles of specific detection of target nucleic acid sequences and subsequent endonuclease-mediated nicking events, wherein the nicking events cause dye groups to dissociate from quenching groups, thereby amplifying the generation of signal events over multiple cycles.

[0103] Some methods of the present invention use multiple single-target-specific hairpin oligonucleotides, universal probes, and nick endonucleases to detect target single-stranded nucleotide sequences in a sample, and then linearly amplify the number of detectable fluorescent signal events over multiple cycles of the reaction. In such methods of the present invention (generally referred to herein as Method 0.5), the method detects target nucleotide sequences in a sample by contacting a target polynucleotide in the sample with an oligonucleotide comprising a hairpin structure (“hairpin”), wherein the target polynucleotide contains a target nucleotide sequence, and the hairpin is characterized by having at least one single-stranded region at a 5' or 3' adjacent position. The single-stranded sequence contains an “A’ sequence” which is complementary to the “A sequence” of the target polynucleotide sequence. The A sequence is part of a target sequence designated as a primary (or first) target sequence. The A’ sequence is adjacent to the B’ sequence of the hairpin. The B’ sequence is complementary to at least a portion of the “B sequence” of the target polynucleotide sequence. The B sequence is continuous with the A sequence and is referred herein as a secondary (or second) target sequence. The B' sequence of the hairpin oligonucleotide hybridizes with its complementary B sequence to form the double-stranded stem structure of the hairpin. The loop structure of the hairpin contains a "C sequence" flanked by B' and B sequences, and includes a sequence containing a nick endonuclease (NE) recognition site. When the target polynucleotide is brought into contact with the hairpin, the hairpin A' sequence hybridizes with the A sequence of the target polynucleotide, serving as the hairpin's foothold. As the B sequence of the secondary target sequence invades the stem structure of the hairpin, the hairpin's B' sequence hybridizes with the secondary target, causing the hairpin's stem-loop structure to open and expose a single-stranded sequence including the hairpin C and B sequences. Subsequently, the oligonucleotide probe of this invention (which contains a C' sequence complementary to the hairpin C sequence) hybridizes with the C sequence, thereby forming an open hairpin and probe complex, which is double-stranded at the hybridization site of the hairpin's C sequence and the probe's C' sequence. The probe is labeled with a quenchable fluorescent dye group attached to or near the 3' or 5' end of the probe, depending on the 5' or 3' sequence orientation of the hairpin A', B', C, and B sequences, or the fluorescent dye group may be located inside the probe. The probe is also labeled with a fluorescent quenching molecule (quencher), also attached to or near the 3' or 5' end of the probe, depending on the 5' or 3' orientation of the hairpin, or the quencher may be located inside the probe, provided the NE site is located between the fluorescent and quenching groups. When the NE comes into contact with the double-stranded hairpin-probe complex, a cleavage reaction produces two probe fragments, one labeled with the fluorescent dye group and the other with the quenching group, allowing the fluorescent dye group to emit a fluorescent signal. After the two probe cleavage fragments dissociate from the C sequence of the opened hairpin, a new labeled probe hybridizes with the C sequence of the hairpin, thus creating a recurring cycle of probe hybridization, cleavage, dissociation, and signal emission.

[0104] Some methods of the present invention use multiple hairpin oligonucleotides, a single universal probe, and a nick endonuclease to detect target single-stranded nucleotide sequences in a sample, and then linearly amplify the number of detectable fluorescent signal events over multiple cycles of the reaction. In this type of method of the present invention (generally referred to herein as Method 1.0), the use of dual hairpins enables the presentation of additional universal probe binding sites. In Method 1.0, the method detects target nucleotide sequences in a sample by contacting a target polynucleotide in the sample with a first oligonucleotide (“HP1”) comprising a hairpin structure, wherein the target polynucleotide contains a target nucleotide sequence, and HP1 is characterized by having at least one single-stranded region at a 5' or 3' adjacent position of the double-stranded stem and single-stranded loop regions. The single-stranded sequence contains an “A’ sequence” which is complementary to the “A sequence” of the target polynucleotide sequence. The A sequence is part of a target sequence designated as a primary (or first) target sequence. The A’ sequence is adjacent to the B’ sequence of the hairpin. The B’ sequence is complementary to at least a portion of the “B sequence” of the target polynucleotide sequence. The B sequence is continuous with the A sequence and is described herein as a secondary (or second) target sequence. The B' sequence of the HP1 oligonucleotide hybridizes with its complementary B sequence to form a double-stranded stem structure of a hairpin. The loop structure of the hairpin contains a "C sequence" flanked by B' and B sequences, and includes a sequence containing a nick endonuclease (NE) recognition site. When a target polynucleotide is brought into contact with HP1, the HP1 A' sequence hybridizes with the A sequence of the target polynucleotide, serving as the foothold of the hairpin. As the B sequence of the secondary target sequence invades the stem structure of HP1, the HP1 B' sequence hybridizes with the secondary target, causing the stem-loop structure of HP1 to open and expose a single-stranded sequence including the HP1 C and HP1 B sequences.

[0105] Method 1.0 also uses a plurality of second hairpin oligonucleotides (HP2) that are specific to HP1 and are characterized by having at least one single-stranded region at a 5' or 3' adjacent position to the double-stranded stem and single-stranded loop regions. The single-stranded sequence contains a "B". S The sequence (i.e., a shortened B' sequence) is complementary to at least one continuous portion of the HP1 B sequence. S The sequence is adjacent to the C' sequence of HP2. The C' sequence is complementary to the C sequence of HP1. The C' sequence is adjacent to the B' sequence. S The sequences are continuous. The C' sequence of the HP2 oligonucleotide forms a hairpin double-stranded stem structure by hybridization with the complementary C sequence. The loop structure of HP2 contains a "B sequence" continuous with the C' sequence and an "A" sequence complementary to at least a portion of the HP A' sequence. S The HP2 sequence (i.e., the shortened A sequence) is flanked by the HP2 C sequence. Upon contact with HP1 containing the target polynucleotide, HP2 B' SThe HP2 sequence hybridizes with the HP1 B sequence, serving as the foothold for HP2. As the HP1 C sequence invades the stem structure of HP2, the HP2 C' sequence hybridizes with the HP1 C sequence, which in turn leads to the HP2 stem-loop structure hybridizing with the complementary sequence on HP1 and exposing a single-stranded sequence including the HP2 C sequence.

[0106] Subsequently, the oligonucleotide probe of the present invention (comprising a C' sequence complementary to the C sequences of HP1 and HP2 and a sequence of the nick endonuclease recognition site) hybridizes with the C sequences of HP1 and HP2 to form an open hairpin-probe complex, which is double-stranded at the hybridization site of the hairpin's C sequence and the probe's C'. The probe is labeled with a quenchable fluorescent dye group attached to or near the 3' or 5' end of the probe, depending on the 5' or 3' sequence orientation of the A', B', C, and B sequences of the hairpin, or the fluorescent dye group is located inside the probe. The probe is also labeled with a fluorescent quenching molecule (quencher), which is also attached to or near the 3' or 5' end of the probe, depending on the 5' or 3' orientation of the hairpin, or the quencher is located inside the probe, provided that the NE site is located between the fluorescent and quenching groups. When the NE comes into contact with the double-stranded hairpin-probe complex, the nick reaction produces two probe fragments, one labeled with the fluorescent dye group and the other labeled with the quencher group, thereby allowing the fluorescent dye group to emit a fluorescent signal. After the two probe cutting fragments dissociate from the C sequence of the open hairpin, a new labeled probe hybridizes with the C sequence of the hairpin, and then the cycle of probe hybridization, cutting, dissociation and signal emission repeats.

[0107] In Method 1.0, the HP2 C' sequence may contain modified thiophosphate residues to prevent unwanted hairpin endonuclease cleavage.

[0108] In the preferred method 1.0, both HP1 and HP2 contain a single-stranded region at the 5' end (5' end protrusion).

[0109] Some methods of this invention use multiple hairpin oligonucleotide pairs, a single universal probe, and two different nick endonucleases to detect target single-stranded nucleotide sequences in a sample, and then linearly amplify the number of detectable fluorescent signal events over multiple cycles of the reaction. In this type of method of the invention (generally referred to herein as method 4.2 or method 4.1), the use of dual hairpins and two different nick endonucleases enables the formation of novel target polynucleotide sites for exponential signal generation via a single universal probe.

[0110] In Method 4.2, the method detects a target nucleotide sequence in a sample by contacting a target polynucleotide in the sample with an oligonucleotide comprising a hairpin structure (“HP1”), wherein the target polynucleotide contains a target nucleotide sequence, and HP1 is characterized by having at least one single-stranded region at a 5' adjacent position between the double-stranded stem and the single-stranded loop region. The single-stranded sequence contains an “A’ sequence” which is complementary to the “A sequence” of the target polynucleotide sequence. The A sequence is part of a target sequence designated as the primary (or first) target sequence. The A’ sequence is adjacent to the B’ sequence of the hairpin. The B’ sequence is complementary to at least a portion of the “B sequence” of the target polynucleotide sequence. The B sequence is continuous with the A sequence and is described herein as a secondary (or second) target sequence, and also contains a sequence of a first nick endonuclease (NE1) recognition site. The B’ sequence of the HP1 oligonucleotide forms the double-stranded stem structure of the hairpin by hybridization with the complementary B sequence. The loop structure of the hairpin contains a “C sequence” which is flanked by the B’ and B sequences. When the target polynucleotide comes into contact with the hairpin, the hairpin A' sequence hybridizes with the A sequence of the target polynucleotide, serving as the hairpin's foothold. As the B sequence of the secondary target sequence invades the hairpin's stem structure, the hairpin's B' sequence hybridizes with the secondary target, which in turn causes the hairpin's stem-loop structure to open and expose single-stranded sequences including the hairpin C and hairpin B sequences.

[0111] Subsequently, the oligonucleotide probe of the present invention (comprising a C' sequence complementary to the C sequence of the hairpin and a sequence of a second nick endonuclease (NE2) recognition site) hybridizes with the C sequence to form an open hairpin-probe complex, which is double-stranded at the hybridization site of the hairpin's C sequence and the probe's C'. The probe is labeled with a quenchable fluorescent dye group attached to or near the 3' or 5' end of the probe, depending on the 5' or 3' sequence orientation of the hairpin's A', B', C, and B sequences, or the fluorescent dye group may be located internally within the probe. The probe is also labeled with a fluorescent quenching molecule (quencher), which is also attached to or near the 3' or 5' end of the probe, depending on the 5' or 3' orientation of the hairpin, or the quencher may be located internally within the probe, provided that the NE site is located between the fluorescent and quenching groups. When NE2 contacts the double-stranded hairpin-probe complex, a nick reaction produces two probe fragments, one labeled with the fluorescent dye group and the other labeled with the quencher group, thereby allowing the fluorescent dye group to emit a fluorescent signal. After the two probe cutting fragments dissociate from the C sequence of the open hairpin, a new labeled probe hybridizes with the C sequence of the hairpin, and then the cycle of probe hybridization, cutting, dissociation and signal emission repeats.

[0112] Method 4.2 also uses a plurality of second hairpin oligonucleotides (HP2) specific to HP1, and is characterized by having at least one single-stranded region at a 3' adjacent position between the double-stranded stem and the single-stranded loop region. The single-stranded sequence contains a "C' sequence" which is complementary to the HP1 C sequence and contains a nucleotide sequence recognition site for the second nick endonuclease (NE2). The C' sequence is adjacent to the B' sequence of HP2. The B' sequence is complementary to the B sequence of HP1. The B' sequence is continuous with the C' sequence. The B' sequence of the HP2 oligonucleotide forms the double-stranded hairpin structure by hybridizing with the complementary B sequence. The loop structure of HP2 contains an "A sequence" which is continuous with the B' sequence and complementary to the HP A' sequence, flanked by the HP2 B sequence. When HP2 comes into contact with HP1 containing the target polynucleotide, the HP2 C' sequence hybridizes with the HP1 C sequence, serving as the foothold for HP2. As the HP1 B sequence invades the stem structure of HP2, the HP2 B' sequence hybridizes with the HP1 B sequence, which in turn causes the stem-loop structure of HP2 to open and expose single-stranded sequences including the HP2 A and B sequences.

[0113] Subsequently, the complexes formed by HP2 and HP1 with the target polynucleotide sequence are contacted with NE1 and NE2 enzymes, resulting in cleavage at the NE1 and NE2 sites. Cleavage at the NE1 site causes the cleaved HP1 B fragment to dissociate from the complex. Cleavage at the NE2 site of the HP2 C' sequence of the HP2 and HP1 complexes with the target polynucleotide causes the short 3' HP2 C' fragment to dissociate; the long HP2 fragment, including the 5' HP2 C' sequence and HP2 B', A, and B sequences, still hybridizes with HP1. Cleavage at both sites on HP1 and HP2 promotes the dissociation of the long HP2 fragment, including the 5' HP2 C' sequence and HP2 B', A, and B sequences, which can then serve as "free" target polynucleotides for further HP1 hybridization. Furthermore, the long HP2 fragment, including the 5' HP2 C' sequence and HP2 B', A, and B sequences, can be replaced by another invading full-length HP2.

[0114] In Method 4.2, these probe hybridizations and HP2 hybridizations, as well as the nicking process, can be repeated to generate fluorescent signals exponentially because more target polynucleotides are produced through the release of the free HP2 fragment. The cycle can continue until the reaction components are depleted.

[0115] In the preferred method 4.2, HP1 contains a single-chain region at the 5' end (5' end protrusion), and HP2 contains a single-chain region at the 3' end (3' end protrusion).

[0116] In Method 4.1, the method detects a target nucleotide sequence in a sample by contacting a target polynucleotide in the sample with an oligonucleotide comprising a hairpin structure (“HP1”), wherein the target polynucleotide contains a target nucleotide sequence, and HP1 is characterized by having at least one single-stranded region at a 5' adjacent position between the double-stranded stem and the single-stranded loop region. The single-stranded sequence contains an “A’ sequence” which is complementary to the “A sequence” of the target polynucleotide sequence. The A sequence is part of a target sequence designated as the primary (or first) target sequence. The A’ sequence is adjacent to the B’ sequence of the hairpin. The B’ sequence is complementary to at least a portion of the “B sequence” of the target polynucleotide sequence. The B sequence is continuous with the A sequence and is referred to herein as the secondary (or second) target sequence. The B’ sequence of the HP1 oligonucleotide forms the double-stranded stem structure of the hairpin by hybridizing with the complementary B sequence. The loop structure of the hairpin contains a “C sequence” which is flanked by the B’ and B sequences. When the target polynucleotide comes into contact with the hairpin, the hairpin A' sequence hybridizes with the A sequence of the target polynucleotide, serving as the hairpin's foothold. As the B sequence of the secondary target sequence invades the hairpin's stem structure, the hairpin's B' sequence hybridizes with the secondary target, which in turn causes the hairpin's stem-loop structure to open and expose single-stranded sequences including the hairpin C and hairpin B sequences.

[0117] Subsequently, the oligonucleotide probe of the present invention (comprising a C' sequence complementary to the C sequence of the hairpin and a sequence of a second nick endonuclease (NE1) recognition site) hybridizes with the C sequence to form an open hairpin-probe complex, which is double-stranded at the hybridization site of the hairpin's C sequence and the probe's C'. The probe is labeled with a quenchable fluorescent dye group attached to or near the 3' or 5' end of the probe, depending on the 5' or 3' sequence orientation of the hairpin's A', B', C, and B sequences, or the fluorescent dye group is located inside the probe. The probe is also labeled with a fluorescent quenching molecule (quencher), which is also attached to or near the 3' or 5' end of the probe, depending on the 5' or 3' orientation of the hairpin, or the quencher is located inside the probe, provided that the NE1 site is located between the fluorescent and quenching groups. When NE1 contacts the double-stranded hairpin-probe complex, a nick reaction produces two probe fragments, one labeled with the fluorescent dye group and the other labeled with the quencher group, thereby allowing the fluorescent dye group to emit a fluorescent signal. After the two probe cutting fragments dissociate from the C sequence of the open hairpin, a new labeled probe hybridizes with the C sequence of the hairpin, and then the cycle of probe hybridization, cutting, dissociation and signal emission repeats.

[0118] Method 4.1 also uses a plurality of HP1-specific second hairpin oligonucleotides (HP2), characterized by having at least one single-stranded region at a 5' adjacent position between the double-stranded stem and the single-stranded loop region. The single-stranded sequence contains a "C' sequence" which is complementary to the HP1 C sequence and contains a nucleotide sequence recognition site for a second nick endonuclease (NE2). The C' sequence is adjacent to the B sequence of HP2. The B sequence is complementary to the B' sequence of HP1. In Method 4.1, HP2 contains a nucleotide sequence recognition site for a second nick endonuclease (NE2) at or between the C' and B sequences. The B sequence of the HP2 oligonucleotide forms a hairpin double-stranded stem structure by hybridizing with the complementary B' sequence. The loop structure of HP2 contains an "A sequence" which is continuous with the B sequence, complementary to the HP A' sequence, and flanked by the HP2 B' sequence. When HP2 comes into contact with HP1 containing the target polynucleotide, the HP2 C' sequence hybridizes with the HP1 C sequence, serving as the foothold for HP2. As the HP1 B' sequence invades the stem structure of HP2, the HP2 B sequence hybridizes with the HP1 B' sequence, which in turn causes the stem-loop structure of HP2 to open and expose the single-stranded sequence including the HP2 B' sequence.

[0119] Subsequently, the complex formed by HP2 and HP1 with the target polynucleotide sequence is contacted with NE1 and NE2 enzymes, cleaving the NE1 and NE2 sites. Cleavage at the NE1 site causes the HP2 C' fragment at the 5' end to dissociate from the complex. Cleavage at the NE2 site of HP2, located at the HP2 B and C' sequence interface, causes the short 3' HP2 C' fragment to dissociate. Cleavage at both sites on HP2 facilitates the replacement of the HP2 fragment containing consecutive B, A, and B' sequences by invasion and hybridization with the new, uncleaved HP2. The 3' B' sequence of the released HP2 fragment can then hybridize with the exposed single-stranded HP1 B sequence, thus revealing additional target polynucleotide A and B sequences.

[0120] In Method 4.1, the hybridization of these probes with HP2 and the nicking process can be repeated, generating a fluorescent signal exponentially because more target polynucleotides are generated by hybridizing additional HP1 to the HP2 fragment, which presents as additional target polynucleotides. The cycle can continue until the reaction components are exhausted.

[0121] In the preferred method 4.1, both HP1 and HP2 contain a single-stranded region (5' end protrusion) at the 5' end.

[0122] In some methods of the present invention, the target polynucleotide comprises a first (primary) target nucleotide sequence (A sequence) and a second (minor) target nucleotide sequence (B sequence), the B sequence being side-joined to the first target nucleotide sequence. In such methods (generally referred to herein as Method 0.0), one or more target polynucleotides are contacted with a plurality of oligonucleotides (each having a hairpin structure), wherein each hairpin oligonucleotide has: (i) a hairpin structure comprising a double-stranded stem region and a single-stranded loop region, wherein: the stem comprises a nucleotide sequence complementary to the B sequence of the target polynucleotide (hairpin B' sequence), which forms a double strand with the complementary sequence (hairpin B sequence); the single-stranded loop region comprises (i) a nucleotide sequence at a nick endonuclease (NE) site (hairpin C sequence); and (ii) a single-stranded major target-specific nucleotide sequence (hairpin A' sequence), located at... The 3' or 5' end of the hairpin stem region includes a nucleotide sequence complementary to the A sequence of the target polynucleotide; the hairpin A' and hairpin B' sequences of one or more hairpin oligonucleotides are hybridized with the A and B sequences of one or more target polynucleotides, respectively, to form a hairpin oligonucleotide-target polynucleotide complex, wherein the hybridization of the hairpin B sequence with the B' sequence of the target polynucleotide opens the stem-loop structure of the hairpin, thereby exposing the single-stranded sequence including the hairpin C and hairpin B sequences; one or more single-stranded oligonucleotide probes (one probe or multiple probes) are then used to hybridize the hairpin oligonucleotides with the target polynucleotide. The exposed single-stranded C and B' sequences of the hairpin structure hybridize to form one or more double-stranded hybrid probe complexes. Each probe comprises a nucleic acid sequence complementary to the hairpin C sequence (C' sequence) and a nucleotide sequence complementary to the hairpin B sequence of the hairpin structure. Each probe is attached with: (i) a quenchable fluorescent group attached to the 3' end, an internal nucleotide position, or the 5' end of the probe; and (ii) a fluorescent quenching molecule (quencher) attached to the 3' end, an internal position, or the 5' end of the probe, wherein the probe cleavage site is located between the fluorescent and quenching groups; allowing one or more NEs at one or more respective NE sites. Under nicking reaction conditions of double-stranded hybrid probe complexes, one or more double-stranded hybrid probe complexes are contacted with one or more nicking endonucleases (NEs), wherein the nicking reaction releases a single-stranded fragment of one or more probes, including a probe fluorophore linker, from each of the one or more hybrid probe complexes containing a quencher, such that: (i) the fluorophore emits a fluorescent signal; and (ii) the probe fragment including the fluorophore dissociates from the loop region, thereby exposing the loop site including the C sequence; and the fluorescence signal of the one or more released probe fragments is detected, wherein the fluorescence signal is correlated with the detection of the target polynucleotide. Steps (C) (i-iii) are repeated until the desired fluorescence signal level is detected.

[0123] The method of this invention can be used to detect target nucleotide sequences in substantially any single-stranded polynucleotide, including, for example, RNA, microRNA (miRNA), single-stranded DNA, or circulating cell-free DNA (cfDNA). Single-stranded DNA targets include single-stranded DNA prepared from double-stranded DNA by means of heat, random cleavage, helicase, or strand displacement by replicative or strand displacement polymerase. In one method of this invention, the target polynucleotide is RNA extracted from a virus, such as variants of SARS-CoV-2, including but not limited to any of the following: N501Y variant, UK (α, B.1.1.7); South Africa (β, B.1.351); Brazil (γ, P.1); India (δ, B.1.617.2); variant B.1.617.2 (δ); and California (ε, B.1.429 / 427).

[0124] In another method of the present invention, a target polynucleotide is used to detect a locus in the tail sequence of a target-specific oligonucleotide primer. One or more target polynucleotides detected in the methods of the present invention may, in some inventions, bind to an antibody or target protein. In the methods of the present invention for multiplex detection of more than one target sequence, the method detects and / or distinguishes mature small RNAs of a specific species from small RNAs of other species in a sample.

[0125] In some methods of the present invention, the method detects very small amounts of target polynucleotides. For example, some methods of the present invention have detection limits (LoD) of 1 to 5 copies / µL, 5 to 15 copies / µL, 15-25 copies / µL, 25-100 copies / µL, 100-200 copies / µL, 200-300 copies / µL, 300-400 copies / µL, 400-500 copies / µL, 500-1000 copies / µL, 1000-1500 copies / µL, or 1500-2000 copies / µL of target polynucleotides.

[0126] The method of this invention is used to detect target nucleotide sequences in samples obtained from any source known to contain, suspected to contain, or suspected of possibly containing target polynucleotides. In one method of this invention, the sample is derived from a biological sample, including samples derived from nasal swabs, oral swabs, pharyngeal swabs, ear swabs, blood or blood components, saliva, urine, or feces. Sample preparation may include a nucleic acid extraction step, or may be prepared using an extraction-free method. In some methods of this invention, the target polynucleotide is prepared using an extraction-free method, such as by heat inactivation or lysis, while extraction-based methods are preferably RNA extraction methods.

[0127] The methods of the present invention can also be used to analyze non-nucleic acid targets. For example, the methods of the present invention can be used to detect the interaction between a binder and a target analyte specifically bound to the binder by: (i) contacting a sample comprising multiple target analytes with multiple binders conjugated to one or more target polynucleotides, under conditions allowing specific binding of the binder to the binder; (ii) removing unbound binders, typically including a washing step; and (ii) performing a method for detecting the target polynucleotide, wherein the detection of a fluorescent signal of one or more released probe fragments indicates specific binding of the binder to the target protein. In some methods of the present invention, the multiple target agents are protein target agents, such as antibodies.

[0128] The A' sequence of the hairpin of the present invention is typically, but not necessarily limited to, 6-24 nucleotides in length. In some hairpin oligonucleotides of the present invention, the A' sequence contains the minimum number of nucleotides required for strand substitution-mediated hairpin opening and subsequent nicking reaction. For example, the A' sequence can be 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, or 30 nucleotides in length.

[0129] The B' sequence and its complementary sequence B of the hairpin of the present invention are typically, but not limited to, 6-24 nucleotides in length. In some hairpin oligonucleotides of the present invention, the A' sequence contains the minimum number of nucleotides required for strand substitution-mediated hairpin opening and subsequent nicking reaction. For example, the B' sequence can be 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, or 30 nucleotides in length.

[0130] The minimum number of nucleotides in the C sequence of the hairpin of the present invention refers to the minimum number of nucleotides required for the hairpin's C sequence to function as a recognition site for a specific nephron endonuclease (NE) used in the method of the present invention when it hybridizes with the C' sequence of the probe of the present invention. Typically, the NE used in the method of the present invention is 3-10 nucleotides in length. In one method of the present invention, the C sequence of the hairpin contains an NE site recognized by Nt.BspQI, while in another method, this site is recognized by Nt.BsmAI, and in other methods of the present invention, it is Nt.CviPII, Nt.BstNBI, Nb.BsrDI, Nb.BtsI, Nt.AlwI, Nb.BbvCI, Nt.BbvCI, Nb.BsmI, or Nb.BssSI.

[0131] The hairpin structure of the present invention may include modifications. For example, in some methods of the present invention, the hairpin structure contains one or more phosphate thioester molecules at or near its 5' or 3' end, or at an internal nucleotide position. In the same or other methods of the present invention, the hairpin structure contains one or more locked nucleic acids (LNAs) at the 5' end, internal nucleotide position, or 3' end of one or more sequence regions of the hairpin. In such methods of the present invention, for example, the A' sequence of the hairpin structure may contain LNAs or other foreign nucleic acids to increase Tm at a specific site. In the same or other methods of the present invention, the hairpin structure contains one or more bridged nucleic acids (BNAs) at the 5' end, internal nucleotide position, or 3' end of one or more sequence regions of the hairpin.

[0132] In some methods of the present invention, one or more sequences of the hairpin or probe may include one or more non-natural nucleic acids, such as peptide nucleic acid (PNA), dNTP / rNTP hybrids, isoguanosine (isoG); isocytidine (isoC), dUTP, rATP, rCTP, rGTP, or rUTP. Furthermore, one or more non-natural nucleic acids may be introduced into the hairpin C sequence of a single-stranded circular region or the hairpin A' sequence of a single-stranded nucleotide sequence.

[0133] In some methods of the present invention, the hairpin structure includes an internal spacer sequence. For example, in one method of the present invention, the hairpin structure includes a spacer region of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length in its A' domain. In another method of the present invention, the hairpin structure includes a spacer region of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length in its B' domain. In another method of the present invention, the hairpin structure includes a spacer region of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length in its B domain. In another method of the present invention, the hairpin structure includes a spacer region of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length in its C' domain. In another method of the present invention, the hairpin structure includes a spacer region of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length in its C domain.

[0134] Similar to the C sequence of the hairpin used in the method of the present invention, the minimum number of nucleotides in the C' sequence of the probe of the present invention refers to the minimum number of nucleotides required for the hairpin's C' sequence to function as the recognition site of the specific ne restriction enzyme (NE) used in the method of the present invention when it hybridizes with the C sequence of the hairpin. Typically, the NE used in the method of the present invention is 3-10 nucleotides long. In one method of the present invention, the C' sequence of the probe contains an NE site recognized by Nt.BspQI, while in another method, this site is recognized by Nt.BsmAI, and in other methods of the present invention, it is Nt.CviPII, Nt.BstNBI, Nb.BsrDI, Nb.BtsI, Nt.AlwI, Nb.BbvCI, Nt.BbvCI, Nb.BsmI, or Nb.BssSI. The following table describes a non-limiting summary of NEs that can be used in the method of the present invention.

[0135] Table 1: Endonuclease sites in the nick

[0136] In some methods of this invention, the probe has at least two distinct NE recognition sites. Such probes are used in multiple applications of the methods of this invention, wherein each distinct NE corresponds to a hairpin that is specific to only one of at least two target sequences in the multiplex analysis. In such methods, the probe (which may be referred to as a dual-enzyme or dual-nicking endonuclease probe) may contain optional X', Y', and Z' sequences for differentially targeting different hairpins in the reaction, or as spacer regions.

[0137] In some methods of the present invention, the quenchable fluorescent group and the quenching molecule are separated by no more than 30 nucleotide positions. Therefore, in the methods of the present invention, the quenchable fluorescent group and the quenching molecule are separated by 1 nucleotide position, 2 nucleotide positions, 3 nucleotide positions, 4 nucleotide positions, 5 nucleotide positions, 6 nucleotide positions, 7 nucleotide positions, 8 nucleotide positions, 9 nucleotide positions, 10 nucleotide positions, 11 nucleotide positions, 12 nucleotide positions, 13 nucleotide positions, 14 nucleotide positions, 15 nucleotide positions, 16 nucleotide positions, 17 nucleotide positions, 18 nucleotide positions, 19 nucleotide positions, 20 nucleotide positions, 21 nucleotide positions, 22 nucleotide positions, 23 nucleotide positions, 24 nucleotide positions, 25 nucleotide positions, 26 nucleotide positions, 27 nucleotide positions, 28 nucleotide positions, 29 nucleotide positions, or 30 nucleotide positions.

[0138] The probes used in the method of this invention can be attached to any fluorescent dye group known to those skilled in the art as suitable for the application of this invention, including, for example, fluorescein (e.g., 5-carboxy-2,7-dichlorofluorescein; 5-carboxyfluorescein (5-FAM); 5-HAT (hydroxytryptamine); 6-HAT; 6-JOE; 6-carboxyfluorescein (6-FAM); FITC); Alexa Fluor (e.g., 350, 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 635, 647, 660, 680, 700, 750); BODIPY TM Fluorescein (e.g., 492 / 515, 493 / 503, 500 / 510, 505 / 515, 530 / 550, 542 / 563, 558 / 568, 564 / 570, 576 / 589, 581 / 591, 630 / 650-X, 650 / 665-X, 665 / 676, FL, FL ATP, Fl-Ceramide, R6GSE, TMR, TMR-X conjugate, TMR-X, SE, TR, TR ATP, TR-X SE), coumarin (e.g., 7-amino-4-methylcoumarin, AMC, AMCA, AMCA-S, AMCA-X, ABQ, CPM methylcoumarin, coumarin phalloidin, hydroxycoumarin, CMFDA, methoxycoumarin), calcein, calcein AM (calcein) AM), calcein blue, calcium dyes (e.g., calcium red, calcium green, calcium orange, calcium fluorescent white), Cascade Blue, Cascade Yellow; Cy TMDyes (e.g., 3, 3.18, 3.5, 5, 5.18, 5.5, 7), cyanGFP, cyclic AMP fluorescent sensor (FiCRhR), fluorescent proteins (e.g., green fluorescent protein (e.g., P. EGFP), blue fluorescent protein (e.g., BFP, EBFP, EBFP2, Azurite, mKalamal), cyan fluorescent protein (e.g., ECFP, Cerulean, CyPet), yellow fluorescent protein (e.g., YFP, Citrine, Venus, YPet), FRET donor / acceptor pairs (e.g., fluorescein / tetramethylrhodamine, lAEDANS / fluorescein, EDANS / dabcyl, fluorescein / fluorescein, BODIPY) TM FL / BODIPY TM FL, fluorescein / QSY7 and QSY9), LysoTracker TM and LysoSensor TM (For example, LysoTracker) TM Blue DND-22, LysoTracker TM Blue-White DPX, LysoTracker TM Yellow HCK-123, LysoTracker TM Green DND-26, LysoTracker TM Red DND-99, LysoSensor TM Blue DND-167, LysoSensor TM Green DND-189, LysoSensor TM Green DND-153, LysoSensor TMYellow / Blue DND-160, LysoSensor Yellow / Blue 10,000 MW dextran), Oregon Green (e.g., 488, 488-X, 500, 514); Rhodamine (e.g., 110, 123, B, B 200, BB, BG, B extra, 5-carboxytetramethylrhodamine (5-TAMRA), 5 GLD, 6-carboxyrhodamine 6G, Lissamine, Lissamine Rhodamine B, phallicidine, phalloidin, Red, Rhod-2, 5-ROX (carboxy-X-rhodamine), sulfonylrhodamine B can C, sulfonylrhodamine G Extra, tetramethylrhodamine (TRITC), WT), Texas Red, Texas Red-X, VIC and other labels, such as those described in U.S. Publication No. 2009 / 0197254), and other labels known to those skilled in the art. Other detectable tags may also be used (see, for example, U.S. Publication No. 2009 / 0197254), as is known to those skilled in the art.

[0139] In some embodiments of the invention, the quenching group is 5IABkFQ, having a broad absorption spectral range from 420 nm to 620 nm and a peak absorption of 531 nm. This quencher can be used with fluorescein and other fluorescent dyes that emit a green to pink spectral range. In some embodiments, the quencher is any Black Hole Quenchers. ® (Available from Biosearch Technologies), Iowa Black ® Any of the quenchers (available from Integrated DNA Technologies), Zen ® Quencher (available from Integrated DNA Technologies), any Onyx ® Quencher (available from Millipore-Sigma) or any ATTO ® Quenching agent (available from ATTO-TEC GmbH).

[0140] The reactions in the methods of the present invention are typically carried out under isothermal conditions, for example, at a temperature of about 20 to 40°C. In one method of the present invention, the reaction is carried out at about 25°C. In another method of the present invention, the reaction is carried out at about 30°C, and in yet another method of the present invention, the reaction is carried out at about 35°C.

[0141] Although the methods of the present invention are described herein as repeating “loops” to amplify signals, they are not hot cycles. For example, the methods of the present invention may repeat 1-500 loops, 1-450 loops, 1-400 loops, 1-350 loops, 1-300 loops, 1-250 loops, 1-200 loops, 1-100 loops, 1-50 loops, 1-45 loops, 1-40 loops, 1-35 loops, 1-30 loops, 1-25 loops, 1-20 loops, 1-15 loops, 1-14 loops, 1-13 loops, 1-12 loops, 1-11 loops, 1-10 loops, 1-9 loops, 1-9 loops, 1-8 loops, 1-7 loops, 1-6 loops, 1-5 loops, 1-4 loops, 1-3 loops, or 2 loops.

[0142] In some methods of the present invention, one or more hairpins and / or probes are conjugated to one or more nanoparticles. For example, the nanoparticles conjugated to the hairpin oligonucleotides of the present invention are polystyrene microspheres or lanthanide nanoparticles, while in another method of the present invention, one or more hairpin oligonucleotides are conjugated to a metal chelating polymer, such as a lanthanide metal chelating polymer.

[0143] Example Example 1: Detection of the SARS-CoV-2 N1 locus SARS-CoV-2 was detected in artificially prepared nasal swab samples containing chemically inactivated and SARS-CoV-2 viral particles (pyrolyzed at 95°C for 5 min) using a linear NICER process. Inactivated viral particles were added directly to the swab samples by placing them in saline solution, and the samples were prepared for analysis at dilutions of 40, 20, 18, 16, 14, 12, 10, 5, and 1 genome copy / μL. Hairpins and probes were tested at a final concentration of 0.25 µM.

[0144] The hairpin nucleotide sequences used in the analysis included a CDC-approved forward primer sequence (SEQ ID NO. 23) specific to the N1 region of the gene encoding the SARS-CoV-2 nucleocapsid protein (N), which is complementary to the A' and B domains of the hairpin structure. Nt.BbvCI was added to the reaction as a nick endonuclease (NE). In these studies, the fluorophore attached to the hairpin hybridization probe was carboxyfluorescein (FAM).

[0145] Two reaction buffer conditions used for NICER were evaluated: (i) 50 mM potassium acetate, 20 mM tris-acetate, 10 mM magnesium acetate, 100 µg / mL recombinant albumin (pH 7.9 @ ​​25°C); and (ii) 10 mM Tris-HCl, 10 mM MgCl2, 50 mM NaCl, 100 µg / mL recombinant albumin (pH 7.9 @ ​​25°C). Higher salt conditions can be used to promote hybridization stability while allowing for enzyme activity. The reaction was performed without a heating cap at 25°C in 20 µL buffer for the entire reaction time. Each imaging cycle was approximately 40 seconds.

[0146] Unlike PCR, each cycle of the NICER process represents an imaging event, rather than a series of thermal cycles linked to imaging. Detection of the fluorescence signal generated when the fluorophore at the probe's cut portion dissociates from the loop sequence is performed using the optics of a standard qPCR instrument.

[0147] By the 10th cycle, signal amplification was observed in all virus dilutions (approximately 6.7 minutes). Figure 8A Some samples showed later cycle signal generation and random loss (Table 2). One of the three negative controls showed amplification products (…). Figure 8B The cycle number data corresponding to Cq are shown in Table 2. The repeat test plots for each limit of detection (LoD) for samples 9, 8, 7, 6, 5, 4, 3, 2, and 1 in Table 2 are shown below. Figure 8C-8K As shown.

[0148] Table 2: N1 Cq Repeated LoD Data

[0149] Given the detection of fluorescence signals in some negative controls, tests were conducted to determine the appropriate concentration of hairpins to minimize oligonucleotide impurities that could cause background noise. Final hairpin concentrations of 50, 25, 10, and 5 nM were tested in negative control samples at a temperature series of 20–30°C. Figures 9A-9D Amplification of the NICER 0.0 control signal was only detected when the reaction was carried out at elevated temperatures using a hairpin concentration of 5 nM, indicating that using a lower concentration of N1 hairpins is advantageous when performing NICER 0.0 at 25°C (standard reaction temperature). However, determining the optimal hairpin concentration at standard temperatures may depend on variables such as the sequence, structure, and size of the hairpins for a specific target.

[0150] Example 2: When the target recognition sequence of the hairpin contains a single nucleotide variant body Time signal amplification is limited To evaluate whether a single nucleotide variant of the target recognition sequence of the NICER hairpin would inhibit target detection, a universal probe NICER method (NICER 0.5) was performed using a SARS-CoV-2 B.1.1.7 - N501Y specific hairpin (“N501 hairpin”) and a SARS-CoV-2 wild-type target. The variant encoding N501Y contains a single nucleotide A / T variation in the gene encoding the surface glycoprotein (S). The N501 hairpin was designed to contain a single nucleotide mismatch at the tail base immediately adjacent to the stem-side junction. The reaction conditions and FAM fluorophore were the same as described in Example 1, but the hairpin was shorter. The same LoD sample as described in Example 1 was used. The test target was the wild-type genome of SARS-CoV-2. It was assumed that the single nucleotide variation was sufficient to disrupt the hairpin opening binding / invasion process, resulting in little or no signal generation. Although a signal was initially observed, the signal amplification pattern was atypical, and signal generation collapsed over multiple cycles, as shown in the generated dome-shaped plot (…). Figure 10A , Figure 10C Two of the three negative controls show dome-shaped signal generation (image). Figure 10B , Figure 10C The composite plot of the N1 and N501 variant hairpins highlights the atypical signal amplification of the N501 hairpin and demonstrates that the NICER method can be used to detect single nucleotide mutations.

[0151] Example 3: Linear NICER using a universal probe ("Method 0.5") The NICER signal amplification was tested using a probe designed to enable non-specific hairpin coupling. Figure 14 A general probe structure is described. In the NICER method using the general probe (“Method 0.5”); Figure 13 In Method 0.5, (i) the A' of a genome target-specific hairpin hybridizes with the A of the target template sequence, and (ii) the hairpin undergoes strand substitution, with the B' of the hairpin hybridizing with the B of the target template sequence; (iii) the C and B of the hairpin become single-stranded, allowing (iv) the C' of the probe to bind to the exposed C portion of the hairpin; (v) a nick endonuclease nicks the probe at the nick site, producing two fragments, each with a similar Tm below 25°C; (vi) the fragments dissociate from the hairpin; and (vii) a new unnicked probe binds to the exposed C portion of the hairpin, and steps (iv)-(vi) are repeated cyclically. Method 0.5 utilizes multiple hairpins specific to different target sequences on the same genome target, as well as single, universal (i.e., non-hairpin specific) probes, to increase signal amplification.

[0152] The NICER amplification using a universal probe was compared with the linear NICER method (“Method 0”) as described in Example 1 above. Reactions were performed on a CFX96 rt-PCR platform at 25°C, 1 s cycles x 250 cycles (excluding imaging); 50 nM template, 50 nM hairpin, and 50 nM probe were used. The results are shown in Figure 33.

[0153] Example 4: Method using internal dyes and quenchers 0.5 The effects of placing quenchers and / or fluorescent dyes inside the probe were investigated. Figure 19 Signal generation between probes with internal quenchers and probes with internal dyes was described. Reactions were performed on a CFX96 rt-PCR platform at 25°C, 1 s cycles x 250 cycles (excluding imaging); using 50 nM template, 50 nM hairpins, and 50 nM probes.

[0154] Using an internal quencher is crucial for significantly reducing the generation of background signals caused by the probe binding directly to the template. Figure 27 The description describes signal amplification of the background signal between a probe with an end quencher and a dye and a probe with an internal quencher and an end dye.

[0155] Example 5: Effect of probe and hairpin concentrations in Method 0.5 The probe was also studied. Figure 21 (see above image) and hairpin density ( Figure 21 The effect of (see figure below) on Method 0.5. Reactions were performed on a CFX96 rt-PCR platform at 25°C; using 5 nM template, 50 nM hairpins, 1 s cycle x 250 cycles (excluding imaging). Experimental conditions for different hairpin concentrations were investigated at 25°C; 5 nM template, 250 nM probe, 1 s cycle x 250 cycles (excluding imaging).

[0156] Example 6: Multiple detection using method 0.5 Multiple detection of two different targets using FAM and ROX fluorescent probes produced distinguishable signals. Figure 23 (See Figure 1-4 above). The reaction was performed on a CFX96 rt-PCR platform with 50 nM template, 50 nM hairpin, and 50 nM probe at 25°C, with ~1 s cycles for 125 cycles (excluding imaging). The control experiment is shown below. Figure 23 As shown in Figure 5-8 below.

[0157] Example 7: The effect of PEG on Method 0.5 and Method 1.0 (see Example 10 below) Method 0.5 was supplemented with 6000 MW polyethylene glycol (PEG) (Figure 39; top). The PEG percentage reflects the final proportion of the reaction mixture. The reaction was performed on a CFX96 rt-PCR platform at 25°C; 50 nM hairpin, 50 nM probe, 1 s cycle x 250 cycles. Different PEG concentrations were also tested at different template concentrations (Figure 39; bottom).

[0158] The 6000 MW PEG and 8000 MW PEG were also evaluated in the Method 1.0 reaction. Figure 25 (See the top figure). The reaction was performed on a CFX96 rt-PCR platform at 25°C, with 250 cycles of 1 s each; 50 nM template, 50 nM hairpin, and 50 nM probe. The effect of 12% 8000 MW PEG on Method 0.5 and Method 1.0 is shown in Figure 40 (bottom figure). The reaction was performed on a CFX96 rt-PCR platform at 25°C, with 250 cycles of 1 s each; 5 nM template, 50 nM / 150 nM hairpin, and 50 nM probe.

[0159] Example 8: Accelerating Linear NICER ("Method 1.0") A dual-spin clip system using a universal probe was also developed, called Accelerated Linear NICER (“Method 1.0”). Figure 16 The modified thiophosphate residues in the second hairpin prevent unwanted endonuclease cleavage. In the first stage of the reaction in Method 1.0, (i) the A' of hairpin 1 (HP1) hybridizes with the A of the target template sequence; (ii) HP1 undergoes strand substitution, and the B' of HP1 hybridizes with the B of the target template sequence; (iii) the C and B of HP1 become single strands; (iv) the C' probe can bind to the exposed C portion of HP1; (v) the cleavage endonuclease cleaves at the recognition site on the probe, producing two fragments, each with a similar Tm below 25°C; (vi) the fragments dissociate from HP1; and (vii) a new uncleaved probe binds to the exposed C portion of HP1, and (iv)-(vi) are repeated as a cycle. In the second stage of the reaction in Method 1.0, (viii) the B' of hairpin 2 (HP2) hybridizes with the B of HP1; (ix) both HP1 and HP2 undergo strand substitution, HP1 dissociates from the template sequence, HP2 hybridizes with HP1, and the C', B, and A sequences of HP2 hybridize with the C, B', and A' sequences of HP1; (x) the original template sequence is used for new HP1 hybridization; (xi) the C' probe can bind to the exposed C portion of HP2; (xii) the nick endonuclease nicks the probe at the nick site, producing two fragments, each with a similar Tm below 25°C, (xiii) the fragments dissociate from HP2; and (xiv) the new unnicked probe binds to the exposed C portion of HP2, and (xi)-(xiii) are repeated cyclically.

[0160] The NICER signal amplification using method 1.0 is compared with that using method 0.5. Figure 26 The reaction was performed on a CFX96 rt-PCR platform at 25°C, 1 s cycle x 250 cycles (excluding imaging); using 50 nM template, 50 nM hairpin, and 50 nM probe.

[0161] Determining the presence of modified thiophosphate residues in the C' region of the second hairpin is important for reducing background signals. Figure 28 The reaction was performed on a CFX96 rt-PCR platform at 25°C, 1 s cycle x 250 cycles (excluding imaging). A representative experimental study of Method 1 without modified hairpin 2 protected the nick site in the C' region, as shown... Figure 28 As shown in the image above, the background signal of the sample without template (gray) is indistinguishable from that of the sample containing template (black). A representative experimental study using method 1 with modified hairpin 2 to protect the incision site in region C' is shown in the image below. Figure 28 There is a clear difference between the background signal of the sample without template (gray) and the sample with template (black).

[0162] Example 9: Exponential NICER ("Method 4.2") A NICER method using a double hairpin, a universal probe, and two restriction enzyme sites / enzymes was also developed for exponential signal generation. The first iteration / implementation of the exponential NICER reaction uses a first hairpin with a 5' protrusion and a second hairpin with a 3' protrusion (“Method 4.2”). Figure 17In the reaction of method 4.2, (i) hairpin 1 (HP1) A' hybridizes with the target template sequence A; (ii) HP1 undergoes strand substitution, and HP1 B' hybridizes with the target template sequence B; (iii) HP1 C and B become single strands; (iv) the C' probe can bind to the exposed C portion of HP1; (v) the first nick endonuclease nicks the probe at the first recognition site, producing two fragments, each with a similar Tm below 25°C; (vi) the fragments dissociate from HP1; (vii) a new unnicked probe binds to the exposed C portion of HP1, and (iv)-(vi) are repeated and cycled; then, (vi) ii) The C' of hairpin 2 (HP2) hybridizes with the exposed C of HP1; (ix) HP2 undergoes strand substitution, and the B' of HP2 hybridizes with the B of HP1; (x) The A and B of HP2 become single strands and are exposed, serving as templates for unhybridized HP1; (xi) The first endonuclease can cleave at the C' recognition site on HP2, allowing the newly uncleaved HP2 to replace the cleaved HP2; and (xii) The second endonuclease can cleave HP1 at the second recognition site in B', causing the short double-stranded sequence generated by the C' of HP1 and the B' of HP2 to dissociate, thereby generating a free target template sequence on the resulting large HP2 fragment. Figure 17 (Right side).

[0163] Example 10: Exponential NICER ("Method 4.1") A second iterative / implementation method for the exponential NICER reaction was also developed, using a first hairpin and a second hairpin (both with 5' protruding ends). Figure 18(Method 4.1). In the reaction of Method 4.1, (i) A' of HP1 hybridizes with A of the target template sequence; (ii) HP1 undergoes strand substitution, and B' of HP1 hybridizes with B of the target template sequence; (iii) C and B of HP1 become single strands; (iv) the C' probe can bind to the exposed C portion of HP1; (v) the first nick endonuclease nicks the recognition site on the probe, producing two fragments, each with a similar Tm below 25°C; (vi) the fragments dissociate from HP1; (vii) a new unnicked probe binds to the exposed C portion of HP1, and (iv)-(vi) are repeated and cycled; then (viii) C' of HP2 hybridizes with B of HP1; (ix) both HP1 and HP2 undergo strand substitution, HP1 dissociates from the template sequence, HP2 hybridizes with HP1, and B and A of HP2 hybridize with B' and A' of HP1; (x) the original template sequence presented on HP2 can be used for new HP... 1. Hybridization; (xi) The C' probe can bind to the exposed C portion of HP2; (xii) The first endonuclease cleaves the probe at the first recognition site, producing two fragments, each with a similar Tm below 25°C, (xiii) the fragments dissociate from HP1; (xiv) A new uncleaved probe binds to the exposed C portion of HP2, and (xi)-(xiii) are repeated and cycled; then (xv) the C' of HP2 can be cleaved by the first and second endonucleases, producing small fragments, each with a similar Tm below 25°C, which can dissociate from HP1; (xvi) A new, uncleaved HP2 can invade and hybridize with HP1, displacing the cleaved HP2; and (xvii) the displaced large HP2 fragment can form a hairpin, or the B' of HP2 can hybridize with the exposed B of HP1, keeping the cleaved HP2 open and providing a new target template sequence for the invasion of unhybridized HP1.

[0164] The feasibility of method 4.2 was demonstrated by agarose gel electrophoresis of the reaction products (Figure 29). Samples were run at 37°C, 1 s cycles x 250 cycles (excluding imaging), then loaded onto 3% agarose gels and electrophoresed on ice at 250 V for 30 min. Template [5 nM], hairpin [50 nM] or [50 nM] / [5 x 10] -4 [nM], probe [150nM]. Agarose gel lanes contained samples run in different reaction buffer formulations to observe the effects of different salt and enzyme compositions and concentrations.

Claims

1. A method for detecting one or more single-stranded target polynucleotides (target polynucleotides) in a sample, wherein each target polynucleotide comprises a first target nucleotide sequence (A sequence) and a second target nucleotide sequence (B sequence) side-joined to the first target nucleotide sequence, the method comprising: (A) Contacting the one or more target polynucleotides with a plurality of hairpin oligonucleotides, wherein each hairpin oligonucleotide comprises: (i) A hairpin structure comprising a double-stranded stem region and a single-stranded loop region (hairpin C sequence), wherein the double-stranded stem region comprises a nucleotide sequence complementary to the B sequence of the target polynucleotide (hairpin B' sequence), which forms a double strand with the complementary sequence (hairpin B sequence); and (ii) A single-stranded nucleotide sequence (hairpin A' sequence) located at the 5' or 3' end of the double-stranded stem region, wherein the single-stranded nucleotide sequence includes a nucleotide sequence complementary to the A sequence of the target polynucleotide; (B) The hairpin A' and hairpin B' sequences of the one or more hairpin oligonucleotides are hybridized with the A and B sequences of the one or more target polynucleotides, respectively, to form a complex of hairpin oligonucleotide and target polynucleotide, wherein the hybridization of the hairpin B' sequence with the target polynucleotide B sequence opens the stem-loop structure of the hairpin, thereby exposing a single-stranded sequence including the hairpin C sequence and the hairpin B sequence. (C) Hybridizing one or more single-stranded oligonucleotide probes (one or more probes) from a plurality of probes to the exposed single-stranded C sequence of the hairpin structure to form one or more double-stranded hybridization probe complexes, each probe comprising a nucleic acid sequence (C' sequence) complementary to the hairpin C sequence and a nucleotide sequence at a nick endonuclease (NE) site, wherein each probe is linked to: (i) a quenchable fluorescent group attached to the 3' end, internal location, or 5' end of the probe; and (ii) A quenching molecule (quencher) attached to the 3' end, internal nucleotide position or 5' end of the probe, wherein the NE site is located between the fluorescent and quenching groups; (D) Under reaction conditions that allow the one or more NEs to nick the double-stranded hybridization probe complex at one or more respective NE sites, the one or more double-stranded hybridization probe complexes are contacted with the one or more NEs, wherein the nicking reaction produces two probe fragments, each fragment dissociating from the hairpin C sequence, thereby releasing the quenchable fluorophore from the quencher and allowing: (i) The fluorescent group emits a fluorescent signal; and (ii) The hairpin C sequence hybridizes with another probe among the plurality of probes; and (E) Detect the fluorescence signal of one or more released probe fragments, wherein the fluorescence signal is correlated with the detection of target polynucleotides.

2. The method of claim 1, further comprising repeating steps (C) and (D) until the desired signal amplification level is reached or until the reactive components are exhausted.

3. A method for detecting one or more single-stranded target polynucleotides (target polynucleotides) in a sample, wherein each target polynucleotide comprises a first target nucleotide sequence (A sequence) and a second target nucleotide sequence (B sequence) side-joined to the first target nucleotide sequence, the method comprising: (A) Contacting the one or more target polynucleotides with a plurality of first hairpin oligonucleotides (HP1), wherein each HP1 comprises: (i) A hairpin structure comprising a double-stranded stem region and a single-stranded loop region (HP1 C sequence), wherein the double-stranded stem region comprises a nucleotide sequence (HP1 B' sequence) complementary to the B sequence of the target polynucleotide, which forms a double strand with the complementary sequence (HP1 B sequence); and (ii) A single-stranded nucleotide sequence (HP1 A' sequence) located at the 5' or 3' end of the double-stranded stem region, wherein the single-stranded nucleotide sequence includes a nucleotide sequence complementary to the A sequence of the target polynucleotide; (B) The HP1 A' and HP1 B' sequences of one or more HP1s are hybridized with the A and B sequences of one or more target polynucleotides, respectively, to form a complex of HP1 and target polynucleotide, wherein the hybridization of the HP1 B' sequence with the B sequence of the target polynucleotide opens the stem-loop structure of HP1, thereby exposing a single-stranded sequence including the HP1 C sequence and the HP1 B sequence. (C) The complex of the HP1 and the target polynucleotide is contacted with a plurality of second hairpin oligonucleotides (HP2), wherein each HP2 comprises: (i) A hairpin structure comprising a double-stranded stem region and a single-stranded loop region, wherein: (a) The double-stranded stem region includes a nucleotide sequence complementary to the HP1 C sequence (HP2 C' sequence), which forms a double strand with the complementary sequence (HP2 C sequence); and (b) The single-stranded circular region includes a nucleotide sequence (HP2 B sequence) complementary to the HP1 B' sequence, the HP2 B sequence being flanked by a nucleotide sequence (HP2 A sequence) complementary to at least one continuous portion of the HP1 A' sequence. S sequence); (ii) Single-stranded nucleotide sequence (HP2 B') S The single-stranded nucleotide sequence is located at the 5' or 3' end of the double-stranded stem region, wherein the single-stranded nucleotide sequence includes a nucleotide sequence complementary to at least one continuous portion of the HP1 B sequence; (D) HP2 B' of one or more HP2 S HP2 C', HP2 B, and HP2 A S The sequences hybridize with the HP1 B, HP1 C, HP1 B' and HP1 A' sequences of the HP1-target polynucleotide complex, respectively, to form HP2 and HP1-target polynucleotide complexes, wherein the hybridization of HP2 with HP1 opens the stem-loop structure of HP2, thereby exposing a single-stranded sequence including the HP2 C sequence. (E) Hybridizing one or more single-stranded oligonucleotide probes (one or more probes) from a plurality of probes with the exposed HP1 C sequence and the exposed single-stranded HP2 sequence of the HP2 and HP1 complex with the target polynucleotide to form one or more double-stranded hybridization probe complexes, each probe comprising a nucleic acid sequence (C' sequence) complementary to the HP1 C sequence and a nucleotide sequence at a nick endonuclease (NE) site, wherein each probe is linked to: (i) a quenchable fluorescent group attached to the 3' end, internal location, or 5' end of the probe; and (ii) A quenching molecule (quencher) attached to the 3' end, internal nucleotide position or 5' end of the probe, wherein the NE site is located between the fluorescent and quenching groups; (F) Under reaction conditions that allow the one or more NEs to nick the double-stranded hybridization probe complex at one or more respective NE sites, the one or more double-stranded hybridization probe complexes are contacted with the one or more NEs, wherein the nicking reaction produces two probe fragments, each fragment dissociating from the hairpin C sequence, thereby releasing the quenchable fluorophore from the quencher and allowing: (i) The fluorescent group emits a fluorescent signal; and (ii) The exposed HP1 and exposed single-stranded HP2 C sequences hybridize with another probe among the plurality of probes; and (G) Detect the fluorescence signal of one or more released probe fragments, wherein the fluorescence signal is correlated with the detection of target polynucleotides.

4. The method of claim 3, wherein the HP2 C' sequence comprises at least one thiophosphate modification.

5. A method for detecting one or more single-stranded target polynucleotides (target polynucleotides) in a sample, wherein each target polynucleotide comprises a first target nucleotide sequence (A sequence) and a second target nucleotide sequence (B sequence) side-joined to the first target nucleotide sequence, the method comprising: (A) Contacting the one or more target polynucleotides with a plurality of first hairpin oligonucleotides (HP1), wherein each HP1 comprises: (i) A hairpin structure comprising a double-stranded stem region and a single-stranded loop region (HP1 C sequence), wherein the double-stranded stem region comprises a nucleotide sequence complementary to the B sequence of the target polynucleotide (HP1 B' sequence), which forms a double strand with the complementary sequence (HP1 B sequence), wherein the HP1 B sequence comprises a nucleotide sequence at a first nick endonuclease (NE1) site; and (ii) A single-stranded nucleotide sequence (HP1 A' sequence) located at the 5' end of the double-stranded stem region, wherein the single-stranded nucleotide sequence includes a nucleotide sequence complementary to the A sequence of the target polynucleotide; (B) The HP1 A' and HP1 B' sequences of one or more HP1s are hybridized with the A and B sequences of one or more target polynucleotides, respectively, to form a complex of HP1 and target polynucleotide, wherein the hybridization of the HP1 B' sequence with the B sequence of the target polynucleotide opens the stem-loop structure of HP1, thereby exposing a single-stranded sequence including the HP1 C sequence and the HP1 B sequence. (C) The complex of the HP1 and the target polynucleotide is contacted with a plurality of second hairpin oligonucleotides (HP2), wherein each HP2 comprises: (i) A hairpin structure comprising a double-stranded stem region and a single-stranded loop region (HP2 A sequence), wherein the double-stranded stem region comprises a nucleotide sequence complementary to the HP1 B sequence (HP2 B' sequence), which forms a double strand with the complementary sequence (HP2 B sequence); and (ii) A single-stranded nucleotide sequence (HP2 C' sequence) located at the 3' end of the double-stranded stem region, wherein the single-stranded nucleotide sequence includes a nucleotide sequence complementary to the HP1 C sequence and a nucleotide sequence at the second NE (NE2) site; (D) The HP2 C' and HP2 B' sequences of one or more HP2s are hybridized with the exposed single-stranded HP1 C and HP1 B sequences of the HP1-target polynucleotide complex, respectively, to form HP2 and HP1-target polynucleotide complexes, wherein the hybridization of HP2 with HP1 opens the stem-loop structure of HP2, thereby exposing single-stranded sequences including HP2 A and HP2 B sequences; (E) Hybridizing one or more single-stranded oligonucleotide probes (one or more probes) from a plurality of probes with an exposed HP1 C sequence to form one or more double-stranded hybridization probe complexes, each probe comprising a nucleic acid sequence (C' sequence) complementary to the HP1 C sequence and a nucleotide sequence at the NE2 site, wherein each probe is linked with: (i) a quenchable fluorescent group attached to the 3' end, internal location, or 5' end of the probe; and (ii) A quenching molecule (quencher) attached to the 3' end, internal nucleotide position or 5' end of the probe, wherein the NE2 site is located between the fluorescent and quenching groups; (F) Contact the one or more double-stranded hybridization probe complexes with the one or more NE2s under reaction conditions that allow the one or more NE2s to perform the following operations: (i) The double-stranded hybridization probe complex is nicked at one or more respective NE2 sites, wherein the nicking reaction produces two probe fragments, each fragment dissociating from the hairpin C sequence, thereby releasing the quenchable fluorophore from the quencher and allowing: (a) The fluorescent group emits a fluorescent signal; and (b) The exposed HP1 and exposed single-stranded HP2 C sequences hybridize with another probe among the plurality of probes; and; (ii) cleaving the NE2 site of the HP2 C' sequence of the complex of the HP2 and HP1 with the target polynucleotide, thereby allowing: (a) Dissociation of the short 3' HP2 C' segment; and (b) A long HP2 fragment comprising the 5' HP2 C' sequence and HP2 B', A and B sequences is replaced by another HP2; (G) Under reaction conditions that allow the one or more NE1s to cleave the NE1 site of the HP1 B sequence of the HP2 and HP1 complex with the target polynucleotide, the HP2 and HP1 complex with the target polynucleotide is contacted with the one or more NE1s, thereby allowing the cleaved HP1 B fragment to dissociate. (H) Detect the fluorescence signal of one or more released probe fragments, wherein the fluorescence signal is correlated with the detection of target polynucleotides.

6. The method of claim 5, wherein the method further comprises: (I) The long HP2 fragment comprising the 5' HP2 C' sequence and HP2 B', A, and B sequences is contacted with another HP1 from a plurality of HP1s, thereby hybridizing the HP1 A' and HP1 B' sequences of the one or more HP1s with the HP2 A and HP2 B sequences, respectively, to form an HP1-HP2 complex, wherein the hybridization of the HP1 B' sequence with the HP2 B sequence opens the stem-loop structure of the HP1, thereby exposing the single-stranded sequences comprising the HP1 C and HP1 B sequences; and (J) Repeat steps (D)-(H) until the desired signal amplification level is reached or until the reactants are exhausted.

7. A method for detecting one or more single-stranded target polynucleotides (target polynucleotides) in a sample, wherein each target polynucleotide comprises a first target nucleotide sequence (A sequence) and a second target nucleotide sequence (B sequence) side-joined to the first target nucleotide sequence, the method comprising: (A) Contacting the one or more target polynucleotides with a plurality of first hairpin oligonucleotides (HP1), wherein each HP1 comprises: (i) A hairpin structure comprising a double-stranded stem region and a single-stranded loop region (HP1 C sequence), wherein the double-stranded stem region comprises a nucleotide sequence (HP1 B' sequence) complementary to the B sequence of the target polynucleotide, which forms a double strand with the complementary sequence (HP1 B sequence); and (ii) A single-stranded nucleotide sequence (HP1 A' sequence) located at the 5' or 3' end of the double-stranded stem region, wherein the single-stranded nucleotide sequence includes a nucleotide sequence complementary to the A sequence of the target polynucleotide; (B) The HP1 A' and HP1 B' sequences of one or more HP1s are hybridized with the A and B sequences of one or more target polynucleotides, respectively, to form a complex of HP1 and target polynucleotide, wherein the hybridization of the HP1 B' sequence with the B sequence of the target polynucleotide opens the stem-loop structure of HP1, thereby exposing a single-stranded sequence including the HP1 C sequence and the HP1 B sequence. (C) The complex of HP1 with the target polynucleotide is contacted with a plurality of second hairpin oligonucleotides (HP2), wherein each HP2 comprises: (i) A hairpin structure comprising a double-stranded stem region and a single-stranded loop region (HP2 A sequence), wherein the double-stranded stem region comprises a nucleotide sequence complementary to the HP1 B sequence (HP2 B sequence), which forms a double strand with the complementary sequence (HP2 B' sequence); (ii) A single-stranded nucleotide sequence (HP2 C' sequence) located at the 5' end of the double-stranded stem region, wherein the single-stranded nucleotide sequence comprises a nucleotide sequence complementary to the HP1 C sequence and a nucleotide sequence at the first nick endonuclease (NE1) site; and (iii) The nucleotide sequence of the second NE (NE2) site, located between the HP2 B sequence and the HP2 C' sequence; (D) The HP2 C', HP2 B' and HP2 A sequences of one or more HP2 are hybridized with the exposed single-stranded HP1 C sequence, HP1 B' sequence and HP1 A' sequence of the HP1-target polynucleotide complex, respectively, so that HP1 dissociates from the target polynucleotide and forms a complex of HP2 and HP1, wherein the hybridization of HP2 and HP1 opens the stem-loop structure of HP2, thereby exposing the single-stranded sequence including the HP2 B' sequence; (E) Hybridizing one or more single-stranded oligonucleotide probes (one or more probes) from a plurality of probes with an exposed HP1 C sequence to form one or more double-stranded hybridization probe complexes, each probe comprising a nucleic acid sequence (C' sequence) complementary to the HP1 C sequence and a nucleotide sequence at the NE1 site, wherein each probe is linked with: (i) a quenchable fluorescent group attached to the 3' end, internal location, or 5' end of the probe; and (ii) A quenching molecule (quencher) attached to the 3' end, an internal nucleotide position, or the 5' end of the probe, wherein the NE1 site is located between the fluorescent and quenching groups; (F) Contact the one or more double-stranded hybridization probe complexes with the one or more NE1s under reaction conditions that allow the one or more NE1s to perform the following operations: (i) The double-stranded hybridization probe complex is nicked at one or more respective NE1 sites, wherein the nicking reaction produces two probe fragments, each fragment dissociating from the hairpin C sequence, thereby releasing the quenchable fluorophore from the quencher and allowing: (a) The fluorescent group emits a fluorescent signal; and (b) The exposed HP1 and exposed single-stranded HP2 C sequences hybridize with another probe among the plurality of probes; and; (ii) cleaving the NE1 site of the HP2 C' sequence of the HP2 and HP1 complex to allow dissociation of the short 5' HP2 C' fragment; and (G) Under reactive conditions that allow the one or more NE2s to cleave the NE2 site located between the HP2 B sequence and the HP2 C' sequence, the complex of HP2 and HP1 is contacted with the one or more NE2s, thereby allowing the 3' HP2 C' fragment of the cleavage to dissociate and form the complex of the cleavage HP2 and HP1. (H) Contact another HP2 from the plurality of HP2s with the HP1 of the HP2 and HP1 complex of the cut, thereby hybridizing the HP2 C', HP2 B' and HP2 A sequences of the one or more HP2s with the exposed single-stranded HP1 C sequence, HP1 B' sequence and HP1 A' sequence, respectively, replacing the cut HP2 including the HP2 B, HP2 A and HPB' sequences; (I) Detect the fluorescence signal of one or more released probe fragments, wherein the fluorescence signal is correlated with the detection of target polynucleotides.

8. The method of claim 7, wherein the HP2 B' sequence of the displaced cut HP2 in step (H) hybridizes with the exposed single-stranded HP1 sequence formed in step (B), thereby allowing hybridization of the HP1 A' and HP1 B' sequences of another HP1 from the plurality of HP1s, wherein hybridization of the HP1 B' sequence with the HP2 B sequence of the HP2 fragment B opens the stem-loop structure of HP1, thereby exposing a single-stranded sequence comprising the HP1 C and HP1 B sequences, the method further comprising repeating steps (D)-(H) until a desired signal amplification level is reached or until the reaction components are exhausted.

9. The method according to any one of claims 1-8, wherein the method is performed under isothermal conditions.

10. The method according to any one of claims 1-8, wherein the method is performed at room temperature.

11. A polynucleotide comprising a hairpin structure and a sequence A', B', C, and B sequentially from 5' to 3' or from 3' to 5', wherein: The A' sequence includes a major target-specific nucleotide sequence; The B' sequence includes a minor target-specific nucleotide sequence; The C sequence includes the nucleotide sequence of the endonuclease recognition site; The B sequence comprises a nucleotide sequence complementary to the B' sequence, and optionally includes a nucleotide sequence representing an endonuclease recognition site; and The B' and B sequences hybridize to form a stem-loop structure, wherein the loop contains a C sequence.

12. The polynucleotide of claim 11, wherein the A' sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides.

13. The polynucleotide of claim 11 or 12, wherein the B' and B sequences comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

14. The polynucleotide according to any one of claims 11-13, wherein the C sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides.

15. The polynucleotide according to any one of claims 11-14, wherein the cleavage endonuclease recognition site is a site required for cleavage by Nt.BspQI, Nt.CviPII, Nt.BstNBI, Nb.BsrDI, Nb.BtsI, Nt.AlwI, Nb.BbvCI, Nt.BbvCI, Nb.BsmI, Nb.BssSI, or Nt.BsmAI.

16. A device comprising a hairpin structure and B's arranged sequentially from 5' to 3' or from 3' to 5'. S Sequence, C' sequence, B sequence, A S Polynucleotides of sequence and C sequence, wherein: (a) The B sequence comprises a nucleotide sequence identical to the secondary target-specific nucleotide sequence; (b) The B' mentioned S The sequence includes a sequence complementary to the B sequence, but comprising fewer nucleotides; (c) The C' sequence comprises a nucleotide sequence of an endonuclease recognition site and at least one modified nucleotide; (d) A S The sequence includes at least one continuous portion of a nucleotide sequence identical to that of the primary target-specific sequence; (e) The C' and C sequences hybridize to form a stem-loop structure, wherein the loop includes the B sequence and the A sequence. S sequence; and (f) The C' sequence includes at least one modified nucleotide that prevents nick endonuclease activity at the nick endonuclease recognition site nucleotide.

17. The polynucleotide of claim 16, wherein the A sequence comprises 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides.

18. The polynucleotide of claim 17 or 18, wherein the B sequence comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

19. The polynucleotide according to any one of claims 16-18, wherein the C and C' sequences comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides.

20. The polynucleotide including the hairpin structure according to claim 16, wherein the modified nucleic acid in (e) is a phosphate thioester modified nucleic acid, peptide nucleic acid (PNA), locked nucleic acid (LNA), dNTP / ribonucleoside triphosphate (rNTP) hybrid, isoguanosine (isoG); isocytidine (isoC), dUTP, rATP, rCTP, rGTP or rUTP.

21. The polynucleotide hairpin structure according to claim 16, wherein the at least one modified nucleic acid is located at: 1-4 nucleotides from the 5' end, at an internal nucleotide position, or at or near the 3' end.

22. A polynucleotide comprising a hairpin structure and a sequence consisting of a C' sequence, a B sequence, an A sequence, and a B' sequence from 5' to 3', wherein: (a) The A sequence includes a major target-specific nucleotide sequence; (b) The B sequence includes a minor target-specific nucleotide sequence and a first nick endonuclease recognition site nucleotide sequence; (c) The B' sequence comprises a nucleotide sequence complementary to the B sequence; (d) The B and B' sequences hybridize to form a stem-loop structure, wherein the loop includes the A sequence; and (e) The C' sequence includes the nucleotide sequence of the second nick endonuclease recognition site.

23. A polynucleotide comprising a hairpin structure and sequences B, A, B', and C' from 5' to 3', wherein: (a) The A sequence includes a major target-specific nucleotide sequence; (b) The B sequence includes a minor target-specific nucleotide sequence; (c) The B' sequence comprises a nucleotide sequence complementary to the B sequence; (d) The B and B' sequences hybridize to form a stem-loop structure, wherein the loop includes the A sequence; (e) The junction of the C' and B sequences includes a second nick endonuclease recognition site nucleotide sequence; and (f) The C' sequence includes the nucleotide sequence of the second nick endonuclease recognition site.

24. The polynucleotide of claim 22 or 23, wherein the A sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides.

25. The polynucleotide of claim 22 or 23, wherein the B and B' sequences comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

26. The polynucleotide of claim 22 or 23, wherein the C' sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides.

27. The polynucleotide hairpin structure according to any one of claims 16-26, wherein the nick endonuclease recognition site, or the first nick endonuclease recognition site and the second nick endonuclease recognition site, are independently sites required for nicking of Nt.BspQI, Nt.CviPII, Nt.BstNBI, Nb.BsrDI, Nb.BtsI, Nt.AlwI, Nb.BbvCI, Nt.BbvCI, Nb.BsmI, Nb.BssSI, or Nt.BsmAI.

28. A linear single-stranded polynucleotide probe, comprising a C' sequence, a dye molecule, and a quencher molecule, wherein: The C' sequence, from 5' to 3' or from 3' to 5', includes: The optional X sequence may consist of 1-8 nucleotides; Nucleotide sequence of the endonuclease recognition site; and The optional Y sequence consists of 1-8 nucleotides.

29. The probe according to claim 28, wherein the nick endonuclease recognition site is a site required for nicking by Nt.BspQI, Nt.CviPII, Nt.BstNBI, Nb.BsrDI, Nb.BtsI, Nt.AlwI, Nb.BbvCI, Nt.BbvCI, Nb.BsmI, Nb.BssSI, or Nt.BsmAI.

30. The probe of claim 28 or claim 29, wherein the probe comprises an X sequence.

31. The probe of claim 30, wherein the X sequence is labeled with a dye, wherein the dye is located inside the X sequence; If the X sequence is located at the 5' end of the C' sequence, then it is located at or near the 5' end of the probe; or If the X sequence is located at the 3' end of the C' sequence, then it is located at or near the 3' end of the probe.

32. The probe according to claim 31, wherein the dye is 6-carboxyfluorescein (6-FAM) or carboxy-X-rhodamine (ROX).

33. The probe according to any one of claims 29-32, wherein the X sequence is labeled with a quenching group, wherein the quenching group is located inside the X sequence; If the X sequence is located at the 5' end of the C' sequence, then it is located at or near the 5' end of the probe; or If the X sequence is located at the 3' end of the C' sequence, then it is located at or near the 3' end of the probe.

34. The probe according to claim 33, wherein the quenching group is a black hole quencher (BHQ).

35. The probe according to any one of claims 28-29, wherein the probe comprises a Y sequence.

36. The probe of claim 30, wherein the Y sequence is labeled with a dye, wherein the dye is located inside the Y sequence; If the Y sequence is located at the 5' end of the C' sequence, then it is located at or near the 5' end of the probe; or If the Y sequence is located at the 3' end of the C' sequence, then it is located at or near the 3' end of the probe.

37. The probe of claim 35, wherein the dye is 6-carboxyfluorescein (6-FAM) or carboxy-X-rhodamine.

38. The probe according to any one of claims 35-37, wherein the Y sequence is labeled with a quenching group, wherein the quenching group: Located inside the Y sequence; If the Y sequence is located at the 5' end of the C' sequence, then it is located at or near the 5' end of the probe; or If the Y sequence is located at the 3' end of the C' sequence, then it is located at or near the 3' end of the probe.

39. The probe according to claim 38, wherein the quenching group is a black hole quencher (BHQ).

40. A linear single-stranded polynucleotide probe, comprising a C' sequence, a dye molecule, and a quencher molecule, wherein: The C' sequence, from 5' to 3' or from 3' to 5', includes: The optional X sequence may consist of 1-4 nucleotides; The first nucleotide sequence of the endonuclease recognition site; The optional Y sequence includes 1-4 nucleotides; The nucleotide sequence of the second nick endonuclease recognition site; and The optional Z sequence consists of 1-4 nucleotides.

41. The probe according to claim 29, wherein the nick endonuclease recognition site is a site required for nicking by Nt.BspQI, Nt.CviPII, Nt.BstNBI, Nb.BsrDI, Nb.BtsI, Nt.AlwI, Nb.BbvCI, Nt.BbvCI, Nb.BsmI, Nb.BssSI, or Nt.BsmAI.

42. The probe of claim 40 or claim 41, wherein the probe comprises an X sequence.

43. The probe of claim 42, wherein the X sequence is labeled with a dye, wherein the dye is located inside the X sequence; If the X sequence is located at the 5' end of the C' sequence, then it is located at or near the 5' end of the probe; or If the X sequence is located at the 3' end of the C' sequence, then it is located at or near the 3' end of the probe.

44. The probe of claim 43, wherein the dye is 6-carboxyfluorescein (6-FAM) or carboxy-X-rhodamine (ROX).

45. The probe according to any one of claims 41-44, wherein the X sequence is labeled with a quenching group, wherein the quenching group: Located inside the X sequence; If the X sequence is located at the 5' end of the C' sequence, then it is located at or near the 5' end of the probe; or If the X sequence is located at the 3' end of the C' sequence, then it is located at or near the 3' end of the probe.

46. ​​The probe according to claim 45, wherein the quenching group is a black hole quencher (BHQ).

47. The probe according to any one of claims 40-41, wherein the probe comprises a Y sequence.

48. The probe of claim 42, wherein the Y sequence is labeled with a dye, wherein the dye is located inside the Y sequence; If the Y sequence is located at the 5' end of the C' sequence, then it is located at or near the 5' end of the probe; or If the Y sequence is located at the 3' end of the C' sequence, then it is located at or near the 3' end of the probe.

49. The probe of claim 47, wherein the dye is 6-carboxyfluorescein (6-FAM) or carboxy-X-rhodamine.

50. The probe according to any one of claims 47-49, wherein the Y sequence is labeled with a quenching group, wherein the quenching group: Located inside the Y sequence; If the Y sequence is located at the 5' end of the C' sequence, then it is located at or near the 5' end of the probe; or If the Y sequence is located at the 3' end of the C' sequence, then it is located at or near the 3' end of the probe.

51. The probe according to claim 50, wherein the quenching group is a black hole quencher (BHQ).

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