A method of detecting a target nucleic acid

Abstract

This application relates to the detection of nucleic acid molecules. Specifically, this application provides a method for detecting one or more target nucleic acids, said method exhibiting improved detection efficiency and enabling polymerases with 5' nuclease activity to achieve higher enzymatic digestion specificity. Furthermore, this application also provides a composition and a kit comprising said composition, said composition and kit being used to implement the method of the present invention.

Description

Technical Field

[0001] This application relates to the detection of nucleic acid molecules. Specifically, this application provides a method for detecting one or more target nucleic acids, which enables the simultaneous detection of multiple target nucleic acid sequences in a sample and enhances the cleavage specificity of polymerase 5' nuclease activity. Furthermore, this application also provides a composition and a kit comprising the composition, which can be used to implement the method of the present invention. Background Technology

[0002] DNA polymerase is an enzyme in living organisms responsible for synthesizing DNA molecules. It forms new DNA strands by pairing nucleotides on a single-stranded DNA template with free deoxyribonucleotide triphosphates (dNTPs). In 1986, Kary Mullis (ColdSpring Harb Symp Quant Biol 1986, 51Pt 1:263-273) invented polymerase chain reaction (PCR) technology, which utilizes the polymerization activity of DNA polymerase to synthesize DNA in vitro. However, the Klenow fragment of E. coli DNA polymerase initially used was not heat-resistant; each time the DNA was denatured by heating, the DNA polymerase would be inactivated and needed to be replenished, a time-consuming, laborious, and error-prone process. In 1988, Saiki et al. (Science 1988, 239(4839):487-491) introduced a thermostable DNA polymerase extracted from Thermus aquaticus (Taq) into PCR technology, solving the problem of having to add a new enzyme after each amplification reaction, and greatly improving the efficiency and stability of PCR amplification.

[0003] Building on this, in 1991, Holland et al. (Proc Natl Acad Sci USA 1991, 88(16): 7276-7280) first confirmed that Taq DNA polymerase possesses 5'→3' nuclease activity, and that this activity, together with polymerization activity, can promote nick translation DNA synthesis. Further, researchers (Nucleic AcidsRes 1993, 21(16): 3761-3766) successfully detected the human cystic fibrosis (CF) gene by utilizing the 5'→3' nuclease activity of Taq DNA polymerase to cleave a fluorescent probe during PCR, thereby emitting a fluorescent signal. Supported by this conclusion, Livak (PCR Methods Appl 1995, 4(6): 357-362) proposed the design scheme of the TaqMan probe, a dual-end labeled fluorescent probe with a fluorophore at the 5' end and a quencher at the 3' end, exhibiting optimal fluorescence signal intensity. This research provided valuable guidance for fluorescent probe design and laid the foundation for innovation in real-time quantitative PCR methods. In 1996, based on these studies, Livak et al. (Genome Res 1996, 6(10): 986-994) combined PCR technology with TaqMan probes to invent the “Real-Time Quantitative PCR” technique. This technique collects fluorescence signals after each PCR cycle and establishes a quantitative relationship between the number of cycles (Ct value) required to reach the exponential amplification phase and the initial concentration of the template. It is easy to see that the TaqMan probe detection system is based on the discovery of the 5'→3' nuclease activity of Taq DNA polymerase, and through continuous improvement and optimization, it has ultimately become a specific, convenient, and widely applicable detection system.

[0004] Corresponding to the 5'→3' nuclease activity of the aforementioned bacterial DNA polymerases is the archaea FEN1 (Flapendonuclease-1) enzyme. In 1999, Lyamichev et al. (J Biol Chem 1999, 274(30): 21387-21394) cloned and purified seven enzymes, including bacterial TaqExo, TaqPol, and TthPol, and archaea AfuFEN, PfuFEN, MjaFEN, and MthFEN, and compared their differences in cleavage activity and substrate specificity. The study found that when the 3'-terminal nucleotide of the upstream oligonucleotide overlapped with the downstream double-stranded DNA by one nucleotide, the cleavage rate of all seven enzymes was significantly increased, but the nuclease cleavage activity of bacterial DNA polymerases was generally lower than that of archaea FEN1 enzymes. In 2000, Lyamichev et al. (Biochemistry 2000, 39(31):9523-9532) again compared the performance of four enzymes derived from archaea and eubacteria (archaea: MthFEN and AfuFEN; eubacteria: TaqPol and TthPol), further confirming that the archaea FEN1 enzyme has superior performance. For example, by comparing the specific and non-specific activities of the four enzymes, they predicted that AfuFEN would have the best signal-to-noise ratio in the "invasive signal amplification reaction." These research results contributed to the establishment of the "Invader" detection technology (NatBiotechnol 1999, 17(3):292-296; Proc Natl Acad Sci USA). 2000, 97(15):8272-8277), This technique uses the archaea FEN1 enzyme and Invader primer. After hybridization with the template, the Invader primer overlaps with the Flap probe by one nucleotide. The FEN1 enzyme specifically cuts the downstream position of the nucleotide in the Flap probe that overlaps with the Invader primer.

[0005] Taq DNA polymerase and archaea FEN1 enzyme have similar structure-specific cleavage activities. However, in Invader assays, only archaea FEN1 enzyme is used with Invader. Even when PCR amplification is combined with Invader technology (the former for amplification, the latter for detection), although the system already contains Taq DNA polymerase with cleavage activity, archaea FEN1 enzyme must still be added separately. This obviously increases the complexity and cost of the assay.

[0006] In target nucleic acid detection methods, various target nucleic acid amplification methods can be combined. If multiplex amplification is used, up to dozens (or even hundreds of) target nucleic acids can be detected simultaneously in the same reaction. Such multiplex amplification typically requires multiple pairs of PCR amplification primers (i.e., target-specific primers) and multiple detection probes. However, in a single system, the presence of a large number of primers and probes significantly increases the difficulty of system establishment. This is because, on the one hand, it is necessary to prevent non-specific extension between primers and between primers and probes, and on the other hand, it is necessary to ensure high-sensitivity detection. Therefore, in a multiplex amplification system, even introducing an additional primer or probe may cause non-specificity, leading to a decrease in the system's detection sensitivity.

[0007] Therefore, it is necessary to develop new nucleic acid detection methods that can be used in various nucleic acid amplification scenarios and can detect nucleic acids with simpler reaction systems and lower detection costs. Summary of the Invention

[0008] To address the aforementioned issues, the applicant conducted extensive research and discovered that the combination of the guide probe, the first oligonucleotide probe, and the polymerase with 5' nuclease activity exhibits high enzyme digestion specificity and digestion speed. Furthermore, compared to existing technologies, the detection method using the combination of the guide probe, the first oligonucleotide probe, and the polymerase with 5' nuclease activity achieved better detection results in both singleton and multiplex detection of target nucleic acids (simultaneous detection of multiple target nucleic acid sequences) (e.g., higher detection signal, shorter detection time, longer amplicon). Moreover, the nucleic acid detection method and the aforementioned composition are adaptable to various nucleic acid amplification methods / platforms and applicable to various detection scenarios (e.g., SNP site detection). Thus, the applicant completed this invention.

[0009] Terminology Definition

[0010] As used herein, the term "oligonucleotide" refers to a molecule containing two or more deoxyribonucleotides or ribonucleotides. The oligonucleotide may contain at least 5-10 nucleotides, at least 10-15 nucleotides, or at least 15-30 nucleotides. The size of an oligonucleotide depends on many factors, as well as its final function and purpose. Oligonucleotides can be produced by a variety of methods, including but not limited to chemical synthesis, DNA replication, and reverse transcription PCR.

[0011] As used herein, the term "adjacent" is intended to mean that two nucleic acid sequences are adjacent to each other without forming a gap. In some embodiments, two adjacent nucleic acid sequences (e.g., region A and region B of the target nucleic acid) are 0 nt apart.

[0012] As used herein, the term “proximate” is intended to mean that two nucleic acid sequences are adjacent to each other, forming a gap. In some preferred embodiments, the two adjacent nucleic acid sequences (e.g., region A and region B of the target nucleic acid) are spaced (or separated) by no more than 20 nt, for example, no more than 15 nt, for example, no more than 10 nt, for example, no more than 5 nt, for example, 4 nt, 3 nt, 2 nt, 1 nt, or 0 nt.

[0013] In this paper, the term "partially overlapping" region refers to a region that hybridizes with two sequences simultaneously. For example, if both the first target-specific sequence and the second target-specific sequence hybridize with the target nucleic acid sequence, and the hybridized region of the target nucleic acid sequence is divided into three distinct regions, then the three regions will be as follows: a region that hybridizes only with the first target-specific sequence; a region that hybridizes only with the second target-specific sequence; and a region that hybridizes with both the first and second target-specific sequences. The region that hybridizes with both the first and second target-specific sequences is the "partially overlapping" region. For example, if both the upstream primer and the first target-specific sequence hybridize with the target nucleic acid sequence, and the hybridized region of the target nucleic acid sequence is divided into three distinct regions, then the three regions will be as follows: a region that hybridizes only with the upstream primer; a region that hybridizes only with the first target-specific sequence; and a region that hybridizes with both the upstream primer and the first target-specific sequence. The region that hybridizes with both the upstream primer and the first target-specific sequence is the "partially overlapping" region.

[0014] As used herein, the term "detectable label" refers to any atom or molecule capable of providing a detectable (preferably quantifiable) signal and capable of being linked to nucleic acids or proteins. These detectable labels can generate signals that can be detected by methods such as fluorescence, radioactivity, colorimetry, specific gravity, X-ray diffraction or absorption, magnetism, enzyme activity, etc.

[0015] As used herein, the term "release" refers to the release of a relatively small nucleic acid fragment from a larger nucleic acid fragment (such as an oligonucleotide) by an enzyme with 5' nuclease activity, such that the released fragment is no longer covalently linked to the rest of the oligonucleotide.

[0016] As used herein, the terms “target nucleic acid sequence,” “target nucleic acid,” and “target sequence” refer to the target nucleic acid sequence to be detected. In this application, the terms “target nucleic acid sequence,” “target nucleic acid,” and “target sequence” have the same meaning and are used interchangeably.

[0017] As used herein, the term "guide probe" is a single-stranded nucleic acid molecule capable of hybridizing with a target nucleic acid, wherein the region of hybridization with the target nucleic acid (e.g., region A) and the region of hybridization with the first oligonucleotide probe (e.g., region B) are close together (e.g., regions A and B are adjacent, adjacent, or partially overlap). In this application, the guide probe comprises a first target-specific sequence comprising a sequence at least partially complementary to a first region of the target nucleic acid. The guide probe is not limited to a second sequence, as long as it contains the first target-specific sequence and the region of hybridization with the target nucleic acid (e.g., region A) and the region of hybridization with the first oligonucleotide probe (e.g., region B) are close together (e.g., regions A and B are adjacent, adjacent, or partially overlap). Therefore, in some embodiments, the guide probe may or may not contain a second sequence downstream of the first target-specific sequence.

[0018] As used herein, the term "first oligonucleotide probe" refers to a single-stranded nucleic acid molecule containing a mediator sequence and a second target-specific sequence in the 5' to 3' direction. In this application, the mediator sequence does not contain a sequence complementary to the target nucleic acid sequence, and the second target-specific sequence contains a sequence complementary to the target nucleic acid sequence. Therefore, under conditions allowing nucleic acid hybridization, annealing, or amplification, the first oligonucleotide probe hybridizes or anneals to the target nucleic acid sequence via the target-specific sequence (i.e., forming a double-stranded structure), and the mediator sequence in the first oligonucleotide probe does not hybridize with the target nucleic acid but remains in a free state (i.e., retains a single-stranded structure).

[0019] As used herein, the terms "target sequence" and "target-specific sequence" refer to a sequence capable of selectively / specifically hybridizing or annealing with a target nucleic acid sequence under conditions allowing nucleic acid hybridization, annealing, or amplification, and which includes a sequence complementary to the target nucleic acid sequence. In this application, the terms "target sequence" and "target-specific sequence" have the same meaning and are used interchangeably. It is readily understood that a target sequence or target-specific sequence is specific to the target nucleic acid sequence. In other words, under conditions allowing nucleic acid hybridization, annealing, or amplification, the target sequence or target-specific sequence hybridizes or anneals only with the specific target nucleic acid sequence and not with other nucleic acid sequences.

[0020] As used herein, the term “mediator sequence” refers to an oligonucleotide sequence in the first oligonucleotide probe that is not complementary to the target nucleic acid sequence and is located upstream (5' end) of the second target-specific sequence.

[0021] As used herein, the term "upstream primer" refers to an oligonucleotide sequence containing a sequence complementary to the target nucleic acid sequence, which is capable of hybridizing (or annealing) or amplifying the target nucleic acid sequence, and which, when hybridizing with the target nucleic acid sequence, is located upstream of the first target-specific sequence or has at least partial overlap with the first target-specific sequence.

[0022] As used herein, the term "complementary" means that two nucleic acid sequences are capable of forming hydrogen bonds with each other according to the base pairing principle (Waston-Crick principle), thereby forming a double helix. In this application, the term "complementary" includes both "substantially complementary" and "completely complementary." As used herein, the term "completely complementary" means that every base in one nucleic acid sequence is capable of pairing with a base in the other nucleic acid strand without mismatches or gaps. As used herein, the term "substantially complementary" means that a majority of the bases in one nucleic acid sequence are capable of pairing with bases in the other nucleic acid strand, allowing for mismatches or gaps (e.g., mismatches or gaps of one or more nucleotides). Typically, under conditions that allow nucleic acid hybridization, annealing, or amplification, two "complementary" (e.g., substantially complementary or completely complementary) nucleic acid sequences will selectively / specifically hybridize or anneal and form a double helix. For example, in this application, the first target-specific sequence of the guide probe and the second target-specific sequence of the first oligonucleotide probe each contain a sequence complementary to (e.g., substantially complementary or completely complementary) the target nucleic acid sequence. Therefore, under conditions allowing nucleic acid hybridization, annealing, or amplification, the target-specific sequences in the guide probe and the first oligonucleotide probe will selectively / specifically hybridize or anneal with the target nucleic acid sequence. Correspondingly, the term "non-complementary" means that the two nucleic acid sequences cannot hybridize or anneal under conditions allowing nucleic acid hybridization, annealing, or amplification, and cannot form a double strand. For example, in this application, the mediator sequence contains a sequence that is not complementary to the target nucleic acid sequence. Therefore, under conditions allowing nucleic acid hybridization, annealing, or amplification, the mediator sequence does not hybridize or anneal with the target nucleic acid sequence, cannot form a double strand, and remains in a free state (i.e., retains a single-stranded structure).

[0023] As used herein, the terms “hybridization” and “annealing” refer to the process by which complementary single-stranded nucleic acid molecules form double-stranded nucleic acids. In this application, “hybridization” and “annealing” have the same meaning and are used interchangeably. Generally, two completely complementary or substantially complementary nucleic acid sequences can hybridize or anneal. The complementarity required for two nucleic acid sequences to hybridize or anneal depends on the hybridization conditions used, particularly the temperature.

[0024] As used herein, “conditions that allow nucleic acid hybridization” has the meaning commonly understood by those skilled in the art and can be determined by conventional methods. For example, two nucleic acid molecules with complementary sequences can hybridize under suitable hybridization conditions. Such hybridization conditions may involve factors such as temperature, pH, composition, and ionic strength of the hybridization buffer, and can be determined based on the lengths and GC contents of the two complementary nucleic acid molecules. For example, low-tightness hybridization conditions may be used when the lengths of the two complementary nucleic acid molecules are relatively short and / or the GC contents are relatively low. High-tightness hybridization conditions may be used when the lengths of the two complementary nucleic acid molecules are relatively long and / or the GC contents are relatively high. Such hybridization conditions are well known to those skilled in the art and can be found, for example, in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); and MLM Anderson, Nucleic Acid Hybridization, Springer-Verlag New York Inc., NY (1999). In this application, “hybridization” and “annealing” have the same meaning and are used interchangeably. Correspondingly, the expressions "conditions that allow nucleic acid hybridization" and "conditions that allow nucleic acid annealing" have the same meaning and can be used interchangeably.

[0025] As used herein, the expression "conditions that allow nucleic acid amplification" has the meaning commonly understood by those skilled in the art as referring to conditions that allow a nucleic acid polymerase (e.g., DNA polymerase) to synthesize another nucleic acid strand using one nucleic acid strand as a template and to form a double helix. Such conditions are well known to those skilled in the art and may involve factors such as temperature, pH, composition, concentration, and ionic strength of the hybridization buffer. Suitable nucleic acid amplification conditions can be determined by conventional methods (see, for example, Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, ColdSpring Harbor Laboratory Press, ColdSpring Harbor, NY (2001)). In the method of the present invention, "conditions that allow nucleic acid amplification" preferably refers to the operating conditions of the nucleic acid polymerase (e.g., DNA polymerase).

[0026] As used herein, the phrase "conditions allowing polymerase to cleave the first oligonucleotide probe" refers to conditions that allow a polymerase with 5' nuclease activity to cleave the first oligonucleotide probe hybridized to the target nucleic acid sequence and release the cleavage product. In the method of the present invention, the conditions allowing cleavage of the first oligonucleotide probe are preferably the operating conditions of the polymerase with 5' nuclease activity. For example, when using a nucleic acid polymerase with 5' nuclease activity, the conditions allowing cleavage of the first oligonucleotide probe can be the operating conditions of the nucleic acid polymerase.

[0027] The operating conditions for various enzymes can be determined by those skilled in the art using conventional methods, and typically involve the following factors: temperature, pH of the buffer solution, composition, concentration, ionic strength, etc. Alternatively, the conditions recommended by the enzyme manufacturer can be used.

[0028] As used herein, the term "upstream" is used to describe the relative positional relationship of two nucleic acid sequences (or two nucleic acid molecules) and has the meaning commonly understood by those skilled in the art. For example, when two nucleic acid sequences (e.g., a guide probe and a first oligonucleotide probe) hybridize with another nucleic acid sequence (e.g., a target nucleic acid), when the 5' to 3' orientation of the guide probe and the first oligonucleotide probe is used as a reference, if the guide probe is located further forward (i.e., closer to the 5' end) than the first oligonucleotide probe, then the guide probe is upstream of the first oligonucleotide probe. In this case, relative to the 5' to 3' orientation of the target nucleic acid, if the guide probe is located further backward (i.e., closer to the 3' end) than the first oligonucleotide probe, then the guide probe is downstream of the target nucleic acid.

[0029] As used herein, the term "upstream" refers to a position closer to the 5' end of a nucleic acid sequence when describing a relative position within the sequence. For example, if a second region within a target nucleic acid sequence is located further forward than the first region (i.e., closer to the 5' end), then the second region is upstream of the first region.

[0030] As used herein, the term "fluorescent probe" refers to an oligonucleotide that carries a fluorescent group and is capable of generating a fluorescent signal. In this application, the fluorescent probe is used as a detection probe.

[0031] As used herein, the term "melting curve analysis" has the meaning commonly understood by those skilled in the art, referring to the method of analyzing the presence or identity of double-stranded nucleic acid molecules by measuring their melting curves, typically used to assess the dissociation characteristics of double-stranded nucleic acid molecules during heating. Methods for performing melting curve analysis are well known to those skilled in the art (see, for example, J Mol Diagn 2009, 11(2):93-101). In this application, the terms "melting curve analysis" and "melting analysis" have the same meaning and are used interchangeably.

[0032] In certain preferred embodiments of this application, melting curve analysis can be performed using oligonucleotide probes labeled with reporter and quencher groups. In short, at ambient temperature, the oligonucleotide probe forms a double strand with its complementary sequence through base pairing. In this case, the reporter group (e.g., fluorophore) and quencher group on the oligonucleotide probe separate from each other, and the quencher group cannot absorb the signal emitted by the reporter group (e.g., fluorescence signal). At this point, the strongest signal (e.g., fluorescence signal) can be detected. As the temperature increases, the two strands of the double strand begin to dissociate (i.e., the detection probe gradually dissociates from its complementary sequence), and the dissociated oligonucleotide probe is in a single-stranded free-coil state. In this case, the reporter group (e.g., fluorophore) and quencher group on the dissociated oligonucleotide probe approach each other, thereby the signal emitted by the reporter group (e.g., fluorophore) (e.g., fluorescence signal) is absorbed by the quencher group. Therefore, as the temperature increases, the detected signal (e.g., fluorescence signal) gradually weakens. When the two strands of the double strand are completely dissociated, all detection probes are in a single-stranded free-coil state. In this case, the signals (e.g., fluorescence signals) emitted by the reporter groups (e.g., fluorescent groups) on all oligonucleotide probes are absorbed by the quenching groups. Therefore, the signals (e.g., fluorescence signals) emitted by the reporter groups (e.g., fluorescent groups) are essentially undetectable. Therefore, by detecting the signals (e.g., fluorescence signals) emitted by the duplex containing the oligonucleotide probe during heating or cooling, the hybridization and dissociation processes of the oligonucleotide probe and its complementary sequence can be observed, forming a curve showing the signal intensity changing with temperature. Further, derivative analysis of the obtained curve yields a curve with the rate of change of signal intensity on the ordinate and temperature on the abscissa (i.e., the melting curve of the duplex). The peaks in this melting curve are the melting peaks, and the corresponding temperatures are the melting points (Tm values) of the duplex. Generally, the higher the degree of matching between the detection probe and the complementary sequence (e.g., fewer mismatched bases and more paired bases), the higher the Tm value of the duplex. Therefore, by detecting the Tm value of the duplex, the presence and identity of the sequence complementary to the detection probe in the duplex can be determined. In this paper, the terms “melting peak”, “melting point” and “Tm value” have the same meaning and are used interchangeably.

[0033] As used herein, the term "SNP (Single Nucleotide Polymorphism)" refers to a nucleic acid sequence polymorphism caused by a single nucleotide variation at the genomic level. The term "SNP site" is a site in the genome that exhibits a single nucleotide polymorphism. In this paper, SNP sites include single sites exhibiting a single nucleotide polymorphism and sites with one or more (e.g., 1, 2, 3, 4, 5, 6, or more) nucleotide insertions or deletions. In this paper, SNP sites are named by their reference number (e.g., rs ID). SNP sites and their types can be queried using the rs ID in public databases, such as the NCBI dbSNP database, the ChinaMAP database, and the JSNP database. As used herein, when referring to the "genotype" of an SNP site, it refers to the collective combination of genes at that SNP site across all homologous chromosomes (usually two homologous chromosomes) of an individual organism.

[0034] As used herein, the term "polymerase" refers to a class of enzymes capable of catalyzing the synthesis of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). "DNA polymerase" is a class of enzymes that use DNA as a template to catalyze the polymerization of substrate dNTP molecules to form new DNA. In eubacteria, native polymerases possess both polymerization activity and 5' nuclease activity, a unique activity found in eubacterial polymerases (e.g., archaea polymerases possess 3' nuclease activity but not 5' nuclease activity). Mutants of polymerases (e.g., naturally occurring or artificially prepared mutants) can retain only 5' nuclease activity. In this document, both native polymerases and said mutants may be used.

[0035] As used in this article, the term "nuclease" refers to a class of enzymes that degrade nucleic acids. Nucleases are hydrolases that act on the PO position of the phosphodiester bond. Nucleases from different sources have different specificities and modes of action. For example, based on the location of their action, nucleases are classified into exonucleases and endonucleases. Further, if an exonuclease hydrolyzes nucleotides one by one starting from the 3' end, it is called a 3'→5' exonuclease (or 3' exonuclease); if an exonuclease hydrolyzes nucleotides one by one starting from the 5' end, it is called a 5'→3' exonuclease (or 5' exonuclease).

[0036] Detection methods

[0037] Therefore, in one aspect, the present invention provides a method for detecting the presence of one or more target nucleic acids in a sample, the method comprising:

[0038] (1) Provide one or more target nucleic acids to be detected, the target nucleic acids comprising a first region and a second region, wherein the first region is located downstream of the second region;

[0039] For each target nucleic acid to be detected, the following are provided:

[0040] At least one guide probe includes a first target-specific sequence comprising a sequence at least partially complementary to a first region of a target nucleic acid; optionally, the guide probe further includes a second sequence downstream of the first target-specific sequence; and

[0041] At least one first oligonucleotide probe includes a mediator sequence and a second target-specific sequence in the 5' to 3' direction, the mediator sequence including a sequence that is not complementary to the target nucleic acid; and the second target-specific sequence including a sequence that is at least partially complementary to a second region of the target nucleic acid;

[0042] Furthermore, under conditions that allow nucleic acid hybridization, the target nucleic acid is contacted with a guide probe and a first oligonucleotide probe;

[0043] (2) Under conditions that allow polymerase to cleave the first oligonucleotide probe, the product of step (1) is contacted with a polymerase having 5' nuclease activity.

[0044] (3) Detect the product of step (2) (e.g., detect whether cleavage occurred in step (2); for example, detect whether one or more cleavage products exist in step (2)) to determine whether one or more target nucleic acids are present in the sample.

[0045] In some embodiments, the nucleotide at the 5' end of the second target-specific sequence is complementary to a second region of the target nucleic acid. In some embodiments, the regions where the guide probe and the first oligonucleotide probe hybridize with the target nucleic acid are close to each other (e.g., adjacent, adjacent to, or partially overlapping).

[0046] In step (1) of the method of the present invention, since the first target-specific sequence contains a sequence that is at least partially complementary to a first region of the target nucleic acid, and the second target-specific sequence contains a sequence that is at least partially complementary to a second region of the target nucleic acid, both the guide probe and the first oligonucleotide probe hybridize with the target nucleic acid when the target nucleic acid is present.

[0047] In step (2) of the method of the present invention, when the target nucleic acid is present, both the guide probe and the first oligonucleotide probe hybridize with the target nucleic acid. Further, since the mediator sequence of the first oligonucleotide probe is not complementary to the target nucleic acid and is in a free state, the first oligonucleotide probe is cleaved by a polymerase with 5' nuclease activity, resulting in the release of a nucleic acid fragment containing the mediator sequence or a portion thereof from the first oligonucleotide probe to form a cleavage product.

[0048] In some preferred embodiments, the regions where the guide probe and the first oligonucleotide probe hybridize with the target nucleic acid are close (e.g., adjacent, contiguous, or partially overlapping), and the 5' terminal nucleotide of the second target-specific sequence of the first oligonucleotide probe is complementary to the target nucleic acid. In this case, a polymerase with 5' nuclease activity becomes enzyme-specific and specifically cleaves the space between the first and second nucleotides at the 5' end of the second target-specific sequence of the first oligonucleotide probe, resulting in the release of a nucleic acid fragment containing the complete mediator sequence and the 5' terminal nucleotide of the second target-specific sequence from the first oligonucleotide probe to form a cleavage product.

[0049] In step (3) of the method of the present invention, the product of step (2) can be detected using any method known in the art. The product of step (2) can be detected by detecting a detectable signal or a change in a detectable signal emitted by a radionuclide, dye, luminescent substance (such as a chemiluminescent substance), or biotin. In some preferred embodiments, in step (3), whether cleavage occurred in step (2) or whether cleavage products are present is detected by detecting the presence of fluorescence (e.g., the presence of a detectable signal emitted by a fluorescent group).

[0050] Guide probe

[0051] As used herein, the term “guide probe” is a single-stranded nucleic acid molecule capable of hybridizing with a target nucleic acid, wherein the region of hybridization with the target nucleic acid (e.g., region A) and the region of hybridization with the target nucleic acid by the first oligonucleotide probe (e.g., region B) are close to each other (e.g., regions A and B are adjacent, adjacent, or partially overlap).

[0052] In this application, the guide probe includes a first target-specific sequence, which comprises a sequence at least partially complementary to a first region of the target nucleic acid. The guide probe is not limited to a second sequence, provided it includes the first target-specific sequence and the region where it hybridizes with the target nucleic acid (e.g., region A) and the region where the first oligonucleotide probe hybridizes with the target nucleic acid (e.g., region B) are close together (e.g., regions A and B are adjacent, adjacent, or partially overlap). Therefore, in some embodiments, the guide probe may or may not include a second sequence downstream of the first target-specific sequence.

[0053] The guide probe does not contain a second sequence.

[0054] In some implementations, the guide probe does not contain a second sequence.

[0055] In some embodiments, the first and second regions of the target nucleic acid are adjacent to each other. As used herein, the term "adjacent" is intended to mean that two nucleic acid sequences are adjacent to each other without forming a gap. In some embodiments, two adjacent nucleic acid sequences (e.g., the first and second regions of the target nucleic acid) are 0 nt apart.

[0056] In some embodiments, upon hybridization with the target nucleic acid, the first target-specific sequence hybridizes to region A of the target nucleic acid, and the second target-specific sequence hybridizes to region B of the target nucleic acid; and, in the target nucleic acid, region A is located downstream of and adjacent to region B. In this document, the first region completely or partially encompasses region A, and the second region completely or partially encompasses region B.

[0057] As used herein, the term “proximate” is intended to mean that two nucleic acid sequences are adjacent to each other, forming a gap. In some preferred embodiments, the two adjacent nucleic acid sequences (e.g., region A and region B of the target nucleic acid) are spaced (or separated) by no more than 20 nt, for example, no more than 15 nt, for example, no more than 10 nt, for example, no more than 5 nt, for example, 4 nt, 3 nt, 2 nt, 1 nt, or 0 nt.

[0058] In some embodiments, region A is separated from region B by 5, 4, 3, 2, 1, or 0 nucleotides. In some preferred embodiments, region A is separated from region B by 2, 1, or 0 nucleotides.

[0059] 3' end

[0060] In this document, the guide probe is not limited to the nucleotides of the 3' portion (e.g., the 3' end). Therefore, in some embodiments, when the guide probe does not contain a second sequence, the nucleotides of the 3' portion (e.g., the 3' end) of the first target-specific sequence may be complementary to or not complementary to the target nucleic acid. In some embodiments, when the guide probe does not contain a second sequence, the first target-specific sequence has a 3'-OH end, or its 3' end is closed. As used herein, the term "3'-terminal nucleotide" refers to the first nucleotide at the 3' end.

[0061] 3' end closed

[0062] In some implementations, the 3' end of the first target-specific sequence is closed.

[0063] In some embodiments, the 3' end of the guide probe is blocked by modifying the 3'-OH of the nucleotide at the 3' end of the first target-specific sequence (e.g., by modifying an amino group or a phosphate group); or by adding a chemical moiety (e.g., biotin, alkyl, or a quencher group) to the 3'-OH of the nucleotide at the 3' end of the first target-specific sequence; or by removing the 3'-OH of the nucleotide at the 3' end of the first target-specific sequence; or by replacing the nucleotide at the 3' end of the first target-specific sequence with a dideoxynucleotide to block the 3' end of the guide probe.

[0064] In some embodiments, when the guide probe does not contain a second sequence, the 3' end of the guide probe is blocked by modifying the 3'-OH of the nucleotide at the 3' end of the first target-specific sequence with an amino group.

[0065] In some implementations, when the guide probe does not contain a second sequence, region A is separated from region B by 0 nucleotides.

[0066] In some implementations, when the guide probe does not contain a second sequence, the nucleotide at the 3' end of the first target-specific sequence is complementary to the target nucleic acid.

[0067] In some embodiments, when the guide probe does not contain a second sequence, the nucleotide at the 3' end of the first target-specific sequence is complementary to the target nucleic acid, and the 3' end is closed (e.g., the nucleotide at the 3' end has an amino group modification); and the region A is separated from the region B by 0 nucleotides.

[0068] In some embodiments, when the guide probe does not contain a second sequence, the 3' end nucleotide of the first target-specific sequence is complementary to the target nucleic acid, and the 3' end is closed (e.g., the 3' end nucleotide has an amino group modification); and the region A is separated from the region B by 2, 1, or 0 nucleotides. In such embodiments, a polymerase derived from eubacteria is used.

[0069] 3' end not closed

[0070] In some embodiments, the first target-specific sequence has a 3'-OH terminus.

[0071] In some implementations, when the guide probe does not contain a second sequence, the nucleotide at the 3' end of the first target-specific sequence is not complementary to the target nucleic acid.

[0072] In some embodiments, when the guide probe does not contain a second sequence, one or more nucleotides of the 3' portion of the first target-specific sequence are not complementary to the target nucleic acid. In some embodiments, when the guide probe does not contain a second sequence, multiple consecutive nucleotides at the 3' end of the first target-specific sequence are not complementary to the target nucleic acid.

[0073] The guide probe contains a second sequence

[0074] In some implementations, the guide probe further includes a second sequence downstream of the first target-specific sequence.

[0075] In some implementations, the first and second regions of the target nucleic acid are adjacent to each other.

[0076] In some embodiments, when hybridizing with the target nucleic acid, the first target-specific sequence hybridizes to region A of the target nucleic acid, and the second target-specific sequence hybridizes to region B of the target nucleic acid; and, in the target nucleic acid, region A and region B are adjacent (e.g., separated by 0 nucleotides).

[0077] In some embodiments, when hybridizing with the target nucleic acid, the second sequence is adjacent to the first target-specific sequence, and the length of the second sequence is 5, 4, 3, 2, or 1 nucleotide. In some preferred embodiments, the length of the second sequence is 3, 2, or 1 nucleotide.

[0078] 3' end

[0079] In this document, the guide probe is not limited to a 3' portion (e.g., a 3' end). Therefore, in some embodiments, when the guide probe also contains a second sequence downstream of the first target-specific sequence, the nucleotides of the 3' portion (e.g., the 3' end) of the second sequence may be complementary or non-complementary to the target nucleic acid. In some embodiments, the second sequence has a 3'-OH end, or its 3' end is closed. As used herein, the term "3'-terminal nucleotide" refers to the first nucleotide at the 3' end.

[0080] 3' end closed

[0081] In some implementations, the 3' end of the second sequence is closed.

[0082] In some embodiments, the 3' end of the guide probe is blocked by modifying the 3'-OH of the nucleotide at the 3' end of the second sequence (e.g., by modifying an amino group or a phosphate group); or by adding a chemical moiety (e.g., biotin, alkyl, or a quencher group) to the 3'-OH of the nucleotide at the 3' end of the second sequence; or by removing the 3'-OH of the nucleotide at the 3' end of the second sequence; or by replacing the nucleotide at the 3' end of the second sequence with a dideoxynucleotide to block the 3' end of the guide probe.

[0083] In some embodiments, when the guide probe contains a second sequence, the 3' end of the guide probe is blocked by modifying the 3'-OH of the nucleotide at the 3' end of the second sequence with an amino group.

[0084] In some embodiments, when the guide probe hybridizes with the target nucleic acid, the second sequence is adjacent to the first target-specific sequence, and the length of the second sequence is 3, 2, or 1 nucleotide.

[0085] In some implementations, when the guide probe contains a second sequence, the nucleotide at the 3' end of the second sequence is complementary to the target nucleic acid.

[0086] In some embodiments, one or more nucleotides of the 3' portion of the second sequence are complementary to the target nucleic acid. In some embodiments, multiple consecutive nucleotides at the 3' end of the second sequence are complementary to the target nucleic acid.

[0087] 3' end not closed

[0088] In some embodiments, the second sequence has a 3'-OH terminus.

[0089] In some embodiments, when the guide probe hybridizes with the target nucleic acid, the second sequence is adjacent to the first target-specific sequence, and the length of the second sequence is 3, 2, or 1 nucleotide.

[0090] In some implementations, when the guide probe contains a second sequence, the nucleotide at the 3' end of the second sequence is not complementary to the target nucleic acid.

[0091] In some embodiments, one or more nucleotides of the 3' portion of the second sequence are not complementary to the target nucleic acid. In some embodiments, multiple consecutive nucleotides at the 3' end of the second sequence are not complementary to the target nucleic acid.

[0092] In some embodiments, when the guide probe includes a second sequence, the nucleotide at the 3' end of the second sequence is not complementary to the target nucleic acid, and the nucleotide has a 3'-OH end; and the region A is separated from the region B by 0 nucleotides, and the length of the second sequence is 1 nucleotide.

[0093] Partially overlapping sequences

[0094] In some embodiments, when hybridizing with the target nucleic acid, the guide probe (e.g., a second sequence of the guide probe), the first oligonucleotide probe (e.g., a second target-specific sequence of the first oligonucleotide probe), and the target nucleic acid have partially overlapping sequences.

[0095] In the text, when hybridizing with the target nucleic acid, the guide probe and the first oligonucleotide probe are aligned in parallel with each other in the same direction, with the guide probe located upstream of the first oligonucleotide probe.

[0096] In the method of this invention, after contacting the target nucleic acid with the guide probe and the first oligonucleotide probe, it is necessary to induce the cleavage of the first oligonucleotide probe to release a fragment containing the complete mediator sequence or a portion thereof. Generally, a polymerase with 5' nuclease activity can be used, utilizing the guide probe or its extension product hybridized with the target nucleic acid, to induce the cleavage of the first oligonucleotide probe hybridized with the target nucleic acid sequence. Specifically, in step (1), when the first oligonucleotide probe contacts the target nucleic acid, its contained second target-specific sequence hybridizes with the target nucleic acid sequence to form a double-stranded structure, while the mediator sequence does not hybridize with the target nucleic acid sequence, maintaining a single-stranded structure. Therefore, a polymerase with 5' nuclease activity can be used to cleave this oligonucleotide containing both double-stranded and single-stranded structures, releasing a fragment with a single-stranded structure.

[0097] In some embodiments of the invention, cleavage of the first oligonucleotide probe can be induced in two ways: (A) in a manner independent of the guide probe extension; and (B) in a manner dependent on the guide probe extension. Specifically, when the guide probe and the first oligonucleotide probe are close together (e.g., adjacent, contiguous, or partially overlapping) and the 3' end of the guide probe is closed, after the guide probe and the first oligonucleotide probe hybridize with the target nucleic acid, a polymerase with 5' nuclease activity can induce cleavage of the 5' portion of the second target-specific sequence of the first oligonucleotide probe (e.g., between the first and second nucleotides at the 5' end), in which case the enzyme will not extend the guide probe (i.e., method A). Conversely, when the guide probe and the first oligonucleotide probe are close together (e.g., adjacent, close to, or partially overlapping each other) and the 3' end of the guide probe is not closed, after the guide probe and the first oligonucleotide probe hybridize with the target nucleic acid, a polymerase with 5' nuclease activity can catalyze the extension of the guide probe. Subsequently, the enzyme with 5' nuclease activity binds to the extension product of the guide probe and cleaves the 5' portion of the second target-specific sequence of the first oligonucleotide probe (e.g., between the first and second nucleotides at the 5' end) (i.e., mode B).

[0098] upstream primer

[0099] In some implementations, for each target nucleic acid to be detected, at least one upstream primer is also provided; wherein the upstream primer contains a sequence complementary to the target nucleic acid; and, when hybridizing with the target nucleic acid, the upstream primer is located upstream of the first target-specific sequence or has a sequence that at least partially overlaps with the first target-specific sequence.

[0100] In some embodiments, after hybridization with the target nucleic acid, the upstream primer is located distal upstream of the first target-specific sequence. In some embodiments, after hybridization with the target nucleic acid, the upstream primer is located adjacent upstream of the first target-specific sequence. In some embodiments, after hybridization with the target nucleic acid, the upstream primer has a partially overlapping sequence with the first target-specific sequence (e.g., the 3' portion of the upstream primer partially overlaps with the 5' portion of the first target-specific sequence). In some embodiments, after hybridization with the target nucleic acid, the upstream primer has a completely overlapping sequence with the first target-specific sequence. In some embodiments, after hybridization with the target nucleic acid, the upstream primer completely contains the first target-specific sequence.

[0101] In some embodiments, in step (1), the target nucleic acid is incubated with the upstream primer and enzyme (e.g., polymerase) under conditions that allow nucleic acid amplification; then, under conditions that allow nucleic acid hybridization, a guide probe and a first oligonucleotide probe are incubated with the amplification product to obtain the product of step (1). In such embodiments, the target nucleic acid is first amplified by primers and enzymes, and then the guide probe and the first oligonucleotide probe are bound to the amplification product for subsequent steps.

[0102] In some embodiments, in step (1), the target nucleic acid is incubated with the upstream primer, enzyme (e.g., polymerase), guide probe, and first oligonucleotide probe under conditions that allow nucleic acid amplification and hybridization to obtain the product of step (1). In such embodiments, the primer and enzyme amplify the target nucleic acid, while the guide probe and first oligonucleotide probe bind to the target nucleic acid and its amplification product for subsequent steps.

[0103] target nucleic acid

[0104] In the method of this invention, the sample can be any sample to be tested. For example, in some preferred embodiments, the sample contains DNA (e.g., genomic DNA or cDNA). In some preferred embodiments, the sample contains RNA (e.g., mRNA). In some preferred embodiments, the sample contains a mixture of nucleic acids (e.g., a mixture of DNA, a mixture of RNA, or a mixture of DNA and RNA).

[0105] In the method of this invention, the target nucleic acid sequence to be detected is not limited by its sequence composition or length. For example, the target nucleic acid sequence may be DNA (e.g., genomic DNA or cDNA) or RNA molecules (e.g., mRNA). Furthermore, the target nucleic acid sequence to be detected may be single-stranded or double-stranded.

[0106] When the sample or target nucleic acid sequence to be detected is mRNA, preferably, a reverse transcription reaction is performed before performing the method of the present invention to obtain cDNA complementary to the mRNA. A detailed description of the reverse transcription reaction can be found, for example, Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001).

[0107] The sample or target nucleic acid sequence to be tested can be obtained from any source, including but not limited to prokaryotes, eukaryotes (e.g., protozoa, parasites, fungi, yeasts, plants, animals including mammals and humans), or viruses (e.g., Herpes virus, HIV, influenza virus, EB virus, hepatitis virus, poliovirus, etc.) or viroids. The sample or target nucleic acid sequence to be tested can also be any form of nucleic acid sequence, such as genomic sequences, artificially isolated or fragmented sequences, synthetic sequences, etc.

[0108] The length of the target nucleic acid and its first and second regions in this invention is not limited. In some embodiments, the length of the first region of the target nucleic acid is 11-150 nt, for example 11-20 nt, 20-30 nt, 30-40 nt, 40-50 nt, 50-60 nt, 60-70 nt, 70-80 nt, 80-90 nt, 90-100 nt, 100-110 nt, 110-120 nt, 120-130 nt, 130-140 nt, or 140-150 nt. In some implementations, the length of the second region of the target nucleic acid is 11-150 nt, for example 11-20 nt, 20-30 nt, 30-40 nt, 40-50 nt, 50-60 nt, 60-70 nt, 70-80 nt, 80-90 nt, 90-100 nt, 100-110 nt, 110-120 nt, 120-130 nt, 130-140 nt, 140-150 nt.

[0109] Therefore, in some embodiments, the sample comprises either DNA, RNA, or a mixture of nucleic acids. In some embodiments, the target nucleic acid is DNA or RNA; and / or, the target nucleic acid is single-stranded or double-stranded. In some embodiments, the target nucleic acid contains one or more SNP sites. In some embodiments, the sample or target nucleic acid is obtained from a source selected from prokaryotes, eukaryotes, viruses, or viroids. In some embodiments, the sample is selected from blood, saliva, urine, feces, cerebrospinal fluid, pleural fluid, breast milk, lymph, sputum, semen, or any combination thereof. In some embodiments, in step (1), 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more target nucleic acids to be detected are provided.

[0110] Polymerase with 5' nuclease activity

[0111] In this application, various polymerases or combinations thereof having 5' nuclease activity can be used to implement the methods of the present invention. The polymerase may be a natural polymerase or a "mutant" having an altered amino acid sequence relative to the natural polymerase, as long as the "mutant" retains or substantially retains (e.g., activity reduced by less than 10%, 20%, or 30%) the activity of the natural polymerase (e.g., 5' nuclease activity). In some preferred embodiments, the polymerase or its mutant comprises both polymerase activity and 5' nuclease activity.

[0112] In some preferred embodiments, the polymerase with 5' nuclease activity is a polymerase with 5' exonuclease activity. In some preferred embodiments, the polymerase with 5' nuclease activity is a polymerase with 5' endonuclease activity. In some preferred embodiments, the polymerase with 5' nuclease activity is a nucleic acid polymerase (e.g., DNA polymerase, particularly a thermostable DNA polymerase) with both 5' nuclease activity (e.g., 5' exonuclease activity and 5' endonuclease activity). In some embodiments, the use of a nucleic acid polymerase with 5' nuclease activity is particularly advantageous because the polymerase can both amplify the target nucleic acid using it as a template and induce cleavage of the first oligonucleotide probe.

[0113] In some preferred embodiments, the DNA polymerase with 5' nuclease activity is a thermostable DNA polymerase, which can be obtained from various eubacterial species, such as *Thermus aquaticus* (Taq), *Thermus thermophiles* (Tth), *Thermus filiformis*, *Thermus flavus*, *Thermococcus literalis*, *Thermus antranildanii*, *Thermus caldophllus*, *Thermus chliarophilus*, *Thermus flavus*, *Thermus igniterrae*, *Thermus lacteus*, *Thermus oshimai*, *Thermus ruber*, *Thermus rubens*, *Thermus scotoductus*, *Thermus silvanus*, *Thermus thermophllus*, *Thermotogamaritima*, *Thermotoga neapolitana*, *Thermosipho africanus*, *Thermococcus litoralis*, *Thermococcus barossi*, *Thermococcus gorgonarius*, *Thermotoga... *Maritima*, *Thermotoganeapolitana*, *Thermosiphoafricanus*, *Pyrococcus woesei*, *Pyrococcus horikoshii*, *Pyrococcus abyssi*, *Pyrodictium occultum*, *Aquifexpyrophilus*, and *Aquifex aeolieus*. In some preferred embodiments, the DNA polymerase having 5' nuclease activity is Taq polymerase.

[0114] In some preferred embodiments, the polymerase in the method of this application may or may not have polymerization activity.

[0115] Therefore, in some embodiments, the polymerase may be selected from any of the following:

[0116] (a) A polymerase with 5' nuclease activity and polymerization activity;

[0117] (b) A polymerase having 5' nuclease activity but lacking polymerization activity (e.g., polymerization activity artificially removed or reduced); or,

[0118] (c) A combination of polymerases as described in (b) and polymerases with polymerization activity.

[0119] In some embodiments, the method of this application does not use a nuclease with 5' nuclease activity. In some embodiments, the method of this application does not use a nuclease derived from archaea. In some embodiments, the method of this application does not use a FEN1 enzyme (e.g., M0645S, NEB).

[0120] Cutting products

[0121] In some embodiments, in step (2), the polymerase having 5' nuclease activity cleaves the first oligonucleotide probe that has hybridized with the target nucleic acid and releases the cleavage product.

[0122] In some embodiments, the polymerase having 5' nuclease activity cleaves the 5' end or 5' portion of the mediator sequence of the first oligonucleotide probe or the second target-specific sequence, and releases a cleavage product containing a portion (5' end portion) of the mediator sequence or the complete mediator sequence.

[0123] In some embodiments, the polymerase having 5' nuclease activity cleaves the 5' portion of the second target-specific sequence of the first oligonucleotide probe (e.g., between the first and second nucleotides at the 5' end).

[0124] In some embodiments, the cleavage product contains the complete mediator subsequence.

[0125] In some embodiments, the cleavage product comprises the complete mediator sequence and a 5' nucleotide of a second target-specific sequence. As used herein, the term "5' nucleotide" refers to the first nucleotide at the 5' end.

[0126] Detection of cut products

[0127] In some implementations, in step (3), the presence of one or more cleavage products from step (2) is detected to determine whether one or more target nucleic acids are present in the sample.

[0128] In some embodiments, melting curve analysis, qPCR-based detection (e.g., using Taqman probes and / or low-background fluorescent probes on a qPCR platform), and / or digital PCR-based detection (e.g., using Taqman probes and / or low-background fluorescent probes on a digital PCR platform) are performed on the cleavage products to determine the presence of one or more target nucleic acids in the sample.

[0129] First oligonucleotide probe

[0130] The first oligonucleotide probe of this application may be equipped with a detectable label to enable the detection of the cleavage product released after cleavage. As used herein, the term "detectable label" refers to any atom or molecule capable of providing a detectable (preferably quantifiable) signal and capable of being linked to a nucleic acid or protein. These detectable labels can generate signals that can be detected by methods such as fluorescence, radioactivity, colorimetry, specific gravity, X-ray diffraction or absorption, magnetism, enzyme activity, etc.

[0131] In some embodiments, the 5' end or 5' portion of the first oligonucleotide probe carries a detectable label. The detectable label may be a directly detectable portion or a binding portion that can be specifically recognized by a second label (e.g., biotin).

[0132] Therefore, in some embodiments, the detectable marker is selected from: dyes, radioactive markers (e.g., 32 P), binding moieties (e.g., biotin), haptens (e.g., digitoxin), luminescent, phosphorescent, or fluorescent moieties (e.g., reporter groups), fluorescent dyes, or any combination thereof.

[0133] In some preferred embodiments, the first oligonucleotide probe is labeled with a reporter group and a quencher group, wherein the reporter group is capable of emitting a signal, and the quencher group is capable of absorbing or quenching the signal emitted by the reporter group; and the signal emitted by the first oligonucleotide probe after being cleaved by the polymerase is different from the signal emitted before being cleaved by the polymerase.

[0134] In some preferred embodiments, in step (3), the presence of one or more target nucleic acids in the sample is determined by detecting whether the reporter group of the first oligonucleotide probe emits a signal after being cleaved by the polymerase to detect whether one or more cleavage products were generated in step (2).

[0135] In some implementations, when not binding to the target nucleic acid, the first oligonucleotide probe is linear or has a hairpin structure. The hairpin structure can be naturally formed or achieved by artificially adding additional bases (e.g., adding a base at the 3' end to complement the 5' end base to form the stem of the hairpin structure; or adding a base at the 5' end to complement the 3' end base to form the stem of the hairpin structure). The number of bases to be added can be determined experimentally to balance hybridization specificity and hybridization efficiency.

[0136] Second oligonucleotide probe

[0137] The method of this application is not limited to a second oligonucleotide probe with a specific structure, as long as it carries a detectable label and at least a portion of its sequence is complementary to all or part of the sequence of the cleavage product. Detection probes known in the art can be applied to the method of this application. Those skilled in the art can select a suitable second oligonucleotide probe based on the detection method (e.g., melting curve analysis, qPCR-based detection, digital PCR-based detection). The design of the second oligonucleotide probe can be referenced, for example, Faltin et al., Clin Chem 2012, 58(11):1546-1556; Huang et al., Proc Natl Acad Sci USA 2022, 119(9):e2110672119.

[0138] In some embodiments, in step (3), at least one second oligonucleotide probe is also provided, and the cleavage product of step (2) is contacted with the second oligonucleotide probe under conditions that allow nucleic acid hybridization;

[0139] In this embodiment, at least a portion of the second oligonucleotide probe is complementary to all or part of the sequence of the cleavage product, and the second oligonucleotide probe is labeled with a detectable tag.

[0140] In some embodiments, the detectable marker is selected from: dyes, radioactive markers (e.g., 32 P), binding moieties (e.g., biotin), haptens (e.g., digitoxin), luminescent, phosphorescent, or fluorescent moieties (e.g., reporter groups), fluorescent dyes, or any combination thereof.

[0141] In some preferred embodiments, the second oligonucleotide probe is labeled with a reporter group and a quencher group, wherein the reporter group is capable of emitting a signal, and the quencher group is capable of absorbing or quenching the signal emitted by the reporter group; and the signal emitted by the second oligonucleotide probe after being cleaved by polymerase is different from the signal emitted before being cleaved by polymerase; or, the signal emitted by the second oligonucleotide probe when hybridizing with its complementary sequence is different from the signal emitted when it does not hybridize with its complementary sequence.

[0142] Second oligonucleotide probe capable of forming its own folded structure

[0143] In some embodiments, the signal emitted by the second oligonucleotide probe after being cleaved by the polymerase is different from the signal emitted before being cleaved by the polymerase; for example, the second oligonucleotide probe generates a detectable signal after being cleaved by the polymerase; and the second oligonucleotide probe is capable of forming a partially overlapping sequence with the cleavage product.

[0144] In some embodiments, the second oligonucleotide probe comprises a complementary sequence and a folded sequence, the complementary sequence being complementary to the cleavage product or a portion thereof, and the folded sequence being capable of forming a partially overlapping sequence with the nucleotide at the 3' end of the cleavage product; for example, the second oligonucleotide probe comprises a first folded sequence, a second sequence, a third folded sequence, and a complementary sequence from 5' to 3', wherein the complementary sequence is complementary to the cleavage product or a portion thereof, and at least a portion of the first folded sequence and at least a portion of the third folded sequence are complementary.

[0145] In some embodiments, the complementary sequence is complementary to the mediator sequence contained in the cleavage product, and the complementary sequence is either complementary to or not complementary to the nucleotide at the 5' end of the second target-specific sequence contained in the cleavage product.

[0146] Second oligonucleotide probe capable of forming stem-loop structure

[0147] In some embodiments, the signal emitted by the second oligonucleotide probe when hybridizing with its complementary sequence is different from the signal emitted when it does not hybridize with its complementary sequence; for example, the second oligonucleotide probe generates a detectable signal after hybridizing with its complementary sequence; wherein the second oligonucleotide probe comprises, from the 3' to the 5' direction: a template sequence and a capture sequence.

[0148] In some embodiments, the second oligonucleotide probe is capable of forming a hairpin structure with a stem and a loop.

[0149] In some implementations, the template sequence is used to form the stem of the hairpin structure when the second oligonucleotide probe forms a hairpin structure.

[0150] In some embodiments, when the second oligonucleotide probe forms a hairpin structure, the capture sequence is contained within the loop of the hairpin structure.

[0151] In some implementations, the capture sequence is complementary to the media subsequence or a portion thereof, or the template sequence is complementary to the media subsequence or a portion thereof.

[0152] MeltArray

[0153] In some embodiments, the capture sequence is complementary to the cleavage product or a portion thereof.

[0154] In some embodiments, under conditions that allow the polymerase to perform an extension reaction, the polymerase uses the capture sequence as a template to extend the cleavage product hybridized to the capture sequence, thereby forming a double strand.

[0155] Mediator Probe

[0156] In some embodiments, the template sequence is complementary to the cleavage product or a portion thereof.

[0157] In some embodiments, under conditions that allow the polymerase to perform an extension reaction, the polymerase uses a template sequence as a template to extend the cleavage product hybridized to the template sequence, thereby forming a double strand.

[0158] Amplification of target nucleic acid

[0159] In some embodiments, in step (1), one or more components (e.g., enzymes, primers or primer pairs for amplifying the target nucleic acid) are also provided, and the one or more components are contacted with the target nucleic acid under conditions that allow nucleic acid amplification. Various methods for amplifying target nucleic acids and the corresponding components involved in amplifying target nucleic acids are disclosed in the art. For example, polymerase chain reaction (PCR), for which the relevant principles can be specifically described in, for example, PCR: Principles and Applications of DNA Amplification, edited by HAErlich, Freeman Press, NY, NY (1992).

[0160] Isothermal amplification

[0161] In some embodiments, isothermal amplification is used to amplify the target nucleic acid. Isothermal amplification is a method of amplifying target nucleic acids using a constant amplification temperature (e.g., a specific temperature between 30°C and 95°C). Unlike standard PCR, isothermal amplification reactions do not involve multiple cycles of denaturation, hybridization, and extension of annealed oligonucleotides. Various types of methods for amplifying target nucleic acid molecules are known in the art, such as nucleic acid sequence-based amplification (NASBA), nickase amplification reaction (NARE), and helicase-dependent amplification (HDA). Therefore, in some embodiments, the isothermal amplification is selected from loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), cross-primer amplification (CPA), nucleic acid sequence-based amplification (NASBA), nickase amplification reaction (NARE), helicase-dependent amplification (HDA), or any combination thereof.

[0162] Loop-mediated isothermal amplification (LAMP)

[0163] In a specific implementation, the isothermal amplification is loop-mediated isothermal amplification (LAMP). The LAMP method uses multiple inner and outer primers (e.g., 2-3 pairs of target-specific primers) and a polymerase with strand displacement activity to amplify the target nucleic acid at a constant temperature (e.g., 60-65°C). For a description of the principle of this method and the required primers, please refer to, for example, Nixon et al., Bimolecular Detection and Quantitation 2014, 2:4-10; Schuler et al., Anal Methods 2016, 8:2750-2755; and Schoepp et al., Sci Transl Med 2017, 9:eaal3693.

[0164] Recombinase polymerase amplification (RPA)

[0165] In a specific implementation, the isothermal amplification is recombinase polymerase amplification (RPA). RPA is a method of amplifying target nucleic acids using multiple upstream primers (e.g., two upstream primers), multiple downstream primers (e.g., three downstream primers), and a polymerase with strand displacement activity at a constant temperature (e.g., 60-65°C). For a description of the principle of this method and the specific primers required, please refer to Piepenburg et al., PLOS Biol 2006, 4(7):e204.

[0166] Cross-primer amplification (CPA) technology

[0167] In a specific implementation, the isothermal amplification is cross-primer amplification (CPA). CPA is a method of amplifying target nucleic acids using an upstream primer (e.g., one upstream primer) and a downstream primer (e.g., one downstream primer) and a polymerase with strand displacement activity at a constant temperature (e.g., 60-65°C). For a description of the principle of this method and the specific primers required, please refer to Xu et al., Sci Rep 2012, 2:246.

[0168] In some embodiments, in step (1), for each target nucleic acid to be detected, at least one downstream primer is provided; wherein the downstream primer contains a sequence complementary to the target nucleic acid sequence; and, when hybridizing with the target nucleic acid sequence, the downstream primer is located downstream of the second target-specific sequence or has a sequence that at least partially overlaps with the second target-specific sequence.

[0169] In some embodiments, in step (1), one or more enzymes (e.g., enzymes with chain displacement activity (e.g., BST enzymes), polymerases) are also provided; then, under conditions that allow nucleic acid hybridization, the target nucleic acid is contacted with the provided upstream primer, downstream primer, guide probe, first oligonucleotide probe and the enzyme.

[0170] In some embodiments, after hybridization with the target nucleic acid, the downstream primer is located distally downstream of the second target-specific sequence. In some embodiments, after hybridization with the target nucleic acid, the downstream primer is located adjacent downstream of the second target-specific sequence. In some embodiments, after hybridization with the target nucleic acid, the downstream primer has a partially overlapping sequence with the second target-specific sequence (e.g., the 3' portion of the second target-specific sequence partially overlaps with the 5' portion of the downstream primer). In some embodiments, after hybridization with the target nucleic acid, the downstream primer has a completely overlapping sequence with the second target-specific sequence. In some embodiments, after hybridization with the target nucleic acid, the downstream primer completely contains the second target-specific sequence.

[0171] In some embodiments, in step (1), the target nucleic acid is mixed with the upstream primer, downstream primer and enzyme under conditions that allow nucleic acid amplification; then, the guide probe and first oligonucleotide probe are mixed with the amplification product under conditions that allow nucleic acid hybridization; or, in step (1), the target nucleic acid is mixed with the upstream primer, downstream primer and enzyme, as well as the guide probe and first oligonucleotide probe, under conditions that allow nucleic acid amplification and hybridization.

[0172] Detection of multiple target nucleic acids

[0173] The method of the present invention can detect two or more target nucleic acids that differ by at least one nucleotide in their respective second regions.

[0174] Therefore, in some embodiments, in step (1) of the method of this application, at least two target nucleic acids to be detected are provided, the target nucleic acids comprising a first region and a second region, wherein the first region is located downstream of the second region; and the second regions of different target nucleic acids have at least one nucleotide difference (e.g., the nucleotides at the 5' end of the second regions of different target nucleic acids are different).

[0175] For each target nucleic acid to be detected, the following are provided:

[0176] At least one guide probe includes a first target-specific sequence comprising a sequence at least partially complementary to a first region of a target nucleic acid; optionally, the guide probe further includes a second sequence downstream of the first target-specific sequence; and

[0177] At least one first oligonucleotide probe includes a mediator sequence and a second target-specific sequence in the 5' to 3' direction, the mediator sequence including a sequence that is not complementary to the target nucleic acid; and the second target-specific sequence including a sequence that is at least partially complementary to a second region of the target nucleic acid;

[0178] Furthermore, under conditions that allow nucleic acid hybridization, the target nucleic acid is contacted with a guide probe and a first oligonucleotide probe.

[0179] In some embodiments, the nucleotide at the 5' end of the second target-specific sequence is complementary to a second region of the target nucleic acid. In some embodiments, the regions where the guide probe and the first oligonucleotide probe hybridize with the target nucleic acid are close to each other (e.g., adjacent, adjacent to, or partially overlapping).

[0180] In the method of this invention, since the second target-specific sequence corresponds one-to-one with the second region of the target nucleic acid, different second target-specific sequences will also be different because the second regions of different target nucleic acids differ by at least one nucleotide. However, it is readily understood that a one-to-one correspondence between the guide probe and the target nucleic acid sequence is not required and can be determined according to the specific detection method. Similarly, whether a one-to-one correspondence between the mediator sequence and the target nucleic acid sequence is also determined according to the specific detection method.

[0181] Therefore, in the method of the present invention, the sequence of the guide probe can be the same or different for different target nucleic acids; and / or, the mediator sequence can be the same or different.

[0182] In some implementations, for each target nucleic acid to be detected, at least one upstream primer is also provided; wherein the upstream primer contains a sequence complementary to the target nucleic acid; and, when hybridizing with the target nucleic acid, the upstream primer is located upstream of the first target-specific sequence or has a sequence that at least partially overlaps with the first target-specific sequence.

[0183] First oligonucleotide probe

[0184] In some embodiments, all first oligonucleotide probes carry different detectable tags. For example, all first oligonucleotide probes carry different reporter groups to achieve detection and / or differentiation of different target nucleic acids. Therefore, the mediator sequence of the first oligonucleotide probes is not limited. In some embodiments, the mediator sequences of all first oligonucleotide probes may be the same or different. In some embodiments, the second target-specific sequences of all first oligonucleotide probes are different from each other.

[0185] In some implementations, the 5' end or 5' portion of the first oligonucleotide probe is marked with a different detectable label.

[0186] In some embodiments, the detectable marker is selected from: dyes, radioactive markers (e.g., 32 P), binding moieties (e.g., biotin), haptens (e.g., digitoxin), luminescent, phosphorescent, or fluorescent moieties (e.g., reporter groups), fluorescent dyes, or any combination thereof.

[0187] In some implementations, the mediator sequences of all the first oligonucleotide probes are different from each other. Therefore, different cleavage products can be detected by means of a second oligonucleotide probe that is complementary to the cleavage product or a portion thereof (e.g., the mediator sequence), thereby enabling the detection and / or differentiation of different target nucleic acids.

[0188] Second oligonucleotide probe capable of forming its own folded structure

[0189] In some embodiments, in step (3), for each cleavage product, at least one second oligonucleotide probe is provided, the second oligonucleotide probe being capable of forming a partially overlapping sequence with a portion of the cleavage product (e.g., the nucleotide at the 3' end); the signal emitted by the second oligonucleotide probe after being cleaved by the polymerase is different from the signal emitted before being cleaved by the polymerase; and different second oligonucleotide probes emit different signals.

[0190] And under conditions that allow nucleic acid hybridization, the cleavage product of step (2) is contacted with the second oligonucleotide probe.

[0191] Second oligonucleotide probe capable of forming stem-loop structure

[0192] MeltArray

[0193] In some embodiments, in step (3), at least one second oligonucleotide probe is also provided, the second oligonucleotide probe comprising, from the 3' to 5' direction, a template sequence, and one or more capture sequences complementary to one or more cleavage products or portions thereof (e.g., the second oligonucleotide probe comprising, from the 3' to 5' direction, a template sequence, and a first capture sequence complementary to the first cleavage product or portions thereof, and a second capture sequence complementary to the second cleavage product or portions thereof); and,

[0194] The second oligonucleotide probe is labeled with a reporter group and a quencher group, wherein the reporter group is capable of emitting a signal, and the quencher group is capable of absorbing or quenching the signal emitted by the reporter group; and the signal emitted by the detection probe when hybridizing with its complementary sequence is different from the signal emitted when it does not hybridize with its complementary sequence.

[0195] And under conditions that allow nucleic acid hybridization, the cleavage product of step (2) is contacted with the second oligonucleotide probe.

[0196] In some preferred embodiments, in step (3), the cleavage product is subjected to melting curve analysis; and based on the results of the melting curve analysis, it is determined whether the multiple target nucleic acid sequences exist in the sample.

[0197] Mediator Probe

[0198] In some embodiments, in step (3), for each cleavage product, at least one second oligonucleotide probe is provided, wherein the second oligonucleotide probe comprises, from the 3' to 5' direction, a template sequence complementary to the cleavage product or a portion thereof, and a capture sequence;

[0199] Furthermore, the signal emitted by the second oligonucleotide probe when it hybridizes with its complementary sequence is different from the signal emitted when it does not hybridize with its complementary sequence; and all second oligonucleotide probes emit different signals.

[0200] And under conditions that allow nucleic acid hybridization, the cleavage product of step (2) is contacted with the second oligonucleotide probe.

[0201] In some embodiments of the present invention, the first and second oligonucleotide probes, as described above, may each independently comprise or consist of naturally occurring nucleotides (e.g., deoxyribonucleotides or ribonucleotides), modified nucleotides, non-natural nucleotides, or any combination thereof. In some preferred embodiments, the first and second oligonucleotide probes each independently comprise or consist of natural nucleotides (e.g., deoxyribonucleotides or ribonucleotides). In some preferred embodiments, the first and second oligonucleotide probes each independently comprise modified nucleotides, such as modified deoxyribonucleotides or ribonucleotides, for example, 5-methylcytosine or 5-hydroxymethylcytosine. In some preferred embodiments, the first and second oligonucleotide probes each independently comprise non-natural nucleotides, such as deoxyhypoxanthine, inosine, 1-(2'-deoxy-β-D-rifuranosyl)-3-nitropyrrole, 5-nitroindole, or locked nucleic acids (LNAs).

[0202] In the method of this invention, neither the first nor the second oligonucleotide probe is limited in length. For example, the lengths of the first and second oligonucleotide probes are independently 11-20 nt, 20-30 nt, 30-40 nt, 40-50 nt, 50-60 nt, 60-70 nt, 70-80 nt, 80-90 nt, 90-100 nt, 100-200 nt, 200-300 nt, 300-400 nt, 400-500 nt, 500-600 nt, 600-700 nt, 700-800 nt, 800-900 nt, or 900-1000 nt. The second target-specific sequence in the first oligonucleotide probe can be of any length, as long as it can specifically hybridize with the target nucleic acid sequence. For example, the length of the second target-specific sequence in the first oligonucleotide probe can be 10-140 nt, such as 10-20 nt, 20-30 nt, 30-40 nt, 40-50 nt, 50-60 nt, 60-70 nt, 70-80 nt, 80-90 nt, 90-100 nt, 100-110 nt, 110-120 nt, 120-130 nt, or 130-140 nt. The mediator sequence in the first oligonucleotide probe can be of any length, as long as it can specifically hybridize with and extend the second oligonucleotide probe. For example, the length of the mediator sequence in the first oligonucleotide probe can be 5-140 nt, such as 5-10 nt, 10-20 nt, 20-30 nt, 30-40 nt, 40-50 nt, 50-60 nt, 60-70 nt, 70-80 nt, 80-90 nt, 90-100 nt, 100-110 nt, 110-120 nt, 120-130 nt, 130-140 nt. In some preferred embodiments, the length of the second target-specific sequence in the first oligonucleotide probe is 10-100 nt (e.g., 10-90 nt, 10-80 nt, 10-50 nt, 10-40 nt, 10-30 nt, 10-20 nt), and the length of the mediator sequence is 5-100 nt (e.g., 10-90 nt, 10-80 nt, 10-50 nt, 10-40 nt, 10-30 nt, 10-20 nt).

[0203] In some preferred embodiments, the first oligonucleotide probe and the second oligonucleotide probe each independently have a 3'-OH terminus. In some preferred embodiments, the 3'-termini of the first oligonucleotide probe and the second oligonucleotide probe are each independently blocked to inhibit their elongation. The 3'-terminus of the nucleic acid (e.g., the first oligonucleotide probe, the second oligonucleotide probe) can be blocked by various methods. For example, the 3'-terminus of the probe can be blocked by modifying the 3'-OH of the last nucleotide of the probe. In some embodiments, the 3'-terminus of the probe can be blocked by adding a chemical moiety (e.g., biotin or an alkyl group) to the 3'-OH of the last nucleotide of the probe. In some embodiments, the 3'-terminus of the probe can be blocked by removing the 3'-OH of the last nucleotide of the probe, or by replacing the last nucleotide with a dideoxynucleotide.

[0204] As described above, the first oligonucleotide probe and the second oligonucleotide probe are each independently labeled with a reporter group and a quencher group, wherein the reporter group is capable of emitting a signal, and the quencher group is capable of absorbing or quenching the signal emitted by the reporter group; and the signal emitted by the detection probe when hybridizing with its complementary sequence is different from the signal emitted when it does not hybridize with its complementary sequence.

[0205] In some preferred embodiments, the first and second oligonucleotide probes are each independently self-quenching probes. In such embodiments, when the first or second oligonucleotide probe does not hybridize with other sequences, the quenching group is located at a position capable of absorbing or quenching the signal of the reporter group (e.g., the quenching group is located adjacent to the reporter group), thereby absorbing or quenching the signal emitted by the reporter group. In this case, the first or second oligonucleotide probe does not emit a signal. Further, when the first or second oligonucleotide probe hybridizes with its complementary sequence, the quenching group is located at a position that cannot absorb or quench the signal of the reporter group (e.g., the quenching group is located far from the reporter group), thereby failing to absorb or quench the signal emitted by the reporter group. In this case, the detection probe emits a signal.

[0206] The design of such self-quenching probes is within the capabilities of those skilled in the art. For example, a reporter group can be labeled at the 5' end of the first or second oligonucleotide probe, while a quenching group can be labeled at the 3' end; alternatively, a reporter group can be labeled at the 3' end of the first or second oligonucleotide probe, while a quenching group is labeled at the 5' end. Thus, when the first or second oligonucleotide probe is present alone, the reporter group and the quenching group approach and interact with each other, causing the signal emitted by the reporter group to be absorbed by the quenching group, thereby preventing the detection probe from emitting a signal. However, when the first or second oligonucleotide probe hybridizes to its complementary sequence, the reporter group and the quenching group separate, preventing the signal emitted by the reporter group from being absorbed by the quenching group, thereby causing the first or second oligonucleotide probe to emit a signal.

[0207] However, it should be understood that the reporter and quencher groups are not necessarily labeled at the ends of the first or second oligonucleotide probe. The reporter and / or quencher groups may also be labeled inside the first or second oligonucleotide probe, provided that the signal emitted by the first or second oligonucleotide probe upon hybridization with its complementary sequence differs from the signal emitted upon hybridization without its complementary sequence. For example, the reporter group may be labeled upstream (or downstream) of the detection probe, and the quencher group downstream (or upstream) of the detection probe, with a sufficient distance between them (e.g., 10-20 nt, 20-30 nt, 30-40 nt, 40-50 nt, 50-60 nt, 60-70 nt, 70-80 nt, or longer). Therefore, when the first or second oligonucleotide probe is present alone, due to the free coiling of the probe molecule or the formation of the probe's secondary structure (e.g., hairpin structure), the reporter group and the quencher group approach and interact with each other, causing the signal emitted by the reporter group to be absorbed by the quencher group, thus preventing the detection probe from emitting a signal. Furthermore, when the detection probe hybridizes with its complementary sequence, the reporter group and the quencher group are separated by a sufficient distance, preventing the signal emitted by the reporter group from being absorbed by the quencher group, thus allowing the detection probe to emit a signal. In some preferred embodiments, the reporter group and the quencher group are separated by a distance of 10-80 nt or longer, for example, 10-20 nt, 20-30 nt, 30-40 nt, 40-50 nt, 50-60 nt, 60-70 nt, or 70-80 nt. In some preferred embodiments, the reporter group and the quencher group are separated by a distance not exceeding 80 nt, 70 nt, 60 nt, 50 nt, 40 nt, 30 nt, or 20 nt. In some preferred embodiments, the reporter group and the quencher group are at least 5 nt apart, at least 10 nt apart, at least 15 nt apart, or at least 20 nt apart.

[0208] Therefore, a reporter group and a quencher group can be labeled at any suitable position on the first or second oligonucleotide probe, provided that the signal emitted by the first or second oligonucleotide probe when hybridizing with its complementary sequence differs from the signal emitted when it does not hybridize with its complementary sequence. However, in some preferred embodiments, at least one of the reporter group and the quencher group is located at the end (e.g., the 5' or 3' end) of the first or second oligonucleotide probe. In some preferred embodiments, one of the reporter group and the quencher group is located at the 5' end of the first or second oligonucleotide probe or at a position 1-10 nt away from the 5' end, and the reporter group and the quencher group are spaced appropriately such that the quencher group can absorb or quench the signal of the reporter group before the first or second oligonucleotide probe hybridizes with its complementary sequence. In some preferred embodiments, one of the reporter group and the quencher group is located at the 3' end of the first or second oligonucleotide probe or at a position 1-10 nt away from the 3' end, and the reporter group and the quencher group are spaced appropriately such that the quencher group can absorb or quench the signal of the reporter group before the first or second oligonucleotide probe hybridizes with its complementary sequence. In some preferred embodiments, the reporter group and the quencher group may be spaced apart as defined above (e.g., 10-80 nt or longer). In some preferred embodiments, one of the reporter group and the quencher group is located at the 5' end of the first oligonucleotide probe or the second oligonucleotide probe, and the other is located at the 3' end.

[0209] In the method of this invention, the reporter group and the quencher group can be any suitable group or molecule known in the art, and specific examples include, but are not limited to, Cy2. TM (506), YO-PRO TM -l(509),YOYO TM -l(509),Calcein(517),FITC(518),FluorX TM (519), Alexa TM (520),Rhodamine 110(520),Oregon Green TM 500(522), Oregon Green TM 488(524),RiboGreen TM (525), Rhodamine Green TM (527),Rhodamine123(529),Magnesium Green TM (531), Calcium GreenTM (533),TO-PRO TM -l(533),TOTOl(533),JOE(548),BODIPY530 / 550(550),Dil(565),BODIPY TMR(568),BODIPY558 / 568(568),BODIPY564 / 570(570),Cy3 TM (570),Alexa TM 546(570),TRITC(572),MagnesiumOrange TM (575),Phycoerythrin R&B(575),Rhodamine Phalloidin(575),CalciumOrange TM (576),PyroninY(580),Rhodamine B(580),TAMRA(582),Rhodamine Red TM (590),Cy3.5 TM (596),ROX(608),Calcium Crimson TM (615),Alexa TM 594(615),Texas Red(615),Nile Red(628),YO-PRO TM -3(631),YOYO TM -3(631),R-phycocyanin(642),C-Phycocyanin(648),TO-PRO TM -3(660),T0T03(660),DiD DilC(5)(665),Cy5 TM(670), Thiadicarbocyanine (671), Cy5.5 (694), HEX (556), TET (536), Biosearch Blue (447), CALFluor Gold 540 (544), CAL Fluor Orange 560 (559), CAL Fluor Red 590 (591), CAL Fluor Red 610 (610), CAL Fluor Red 635 (637), FAM (520), Fluorescein (520), Fluorescein-C3 (520), Pulsar 650 (566), Quasar 570 (667), Quasar 670 (705), and Quasar 705 (610). The numbers in parentheses indicate the maximum emission wavelength in nm.

[0210] Furthermore, various suitable pairings of reporter and quencher groups are known in the art; see, for example, Pesce et al., editors, Fluorescence Spectroscopy (Marcel Dekker, New York, 1971); White et al., Fluorescence Analysis: A Practical Approach (Marcel Dekker, New York, 1970); Berlman, Handbook of Fluorescence Spectra of Aromatic Molecules, 2nd Edition (Academic Press, New York, 1971); Griffiths, Color and Constitution of Oiganic Molecules (Academic Press, New York, 1976); Bishop, editor, Indicators (Peigamon Press, Oxford, 1972); Haugland, Handbook of Fluorescent Probes and Research Chemicals (Molecular Probes, Eugene, 1992); Pringsheim, Fluorescence and Phosphorescence (Interscience Publishers, New York). York, 1949; Haugland, RP, Handbook of Fluorescent Probes and Research Chemicals, 6th Edition (Molecular Probes, Eugene, Oreg., 1996); U.S. Patents 3,996,345 and 4,351,760.

[0211] In some preferred embodiments, the reporter group is a fluorescent group. In such embodiments, the signal emitted by the reporter group is fluorescence, and the quencher group is a molecule or group capable of absorbing / quenching the fluorescence (e.g., another fluorescent molecule capable of absorbing the fluorescence, or a quencher capable of quenching the fluorescence). In some preferred embodiments, the fluorescent group includes, but is not limited to, various fluorescent molecules, such as ALEX-350, FAM, VIC, TET, and CAL. Gold 540, JOE, HEX, CAL Fluor Orange 560, TAMRA, CAL Fluor Red 590, ROX, CAL Fluor Red 610, TEXAS RED, CAL Fluor Red 635, Quasar 670, CY3, CY5, CY5.5, Quasar 705, etc. In some preferred embodiments, the quenching group includes, but is not limited to, various quenching agents, such as DABCYL, BHQ (e.g., BHQ-1 or BHQ-2), ECLIPSE, and / or TAMRA, etc.

[0212] In some embodiments, the first oligonucleotide probe is linear or has a hairpin structure. In some embodiments, the hairpin structure can be naturally occurring or can be achieved by artificially adding additional bases.

[0213] In some embodiments of the invention, the guide probe as described above comprises or consists of naturally occurring nucleotides (e.g., deoxyribonucleotides or ribonucleotides), modified nucleotides, non-natural nucleotides, or any combination thereof. In some preferred embodiments, the guide probes each independently comprise or consist of natural nucleotides (e.g., deoxyribonucleotides or ribonucleotides). In some preferred embodiments, the guide probes comprise modified nucleotides, such as modified deoxyribonucleotides or ribonucleotides, such as 5-methylcytosine or 5-hydroxymethylcytosine. In some preferred embodiments, the guide probes comprise non-natural nucleotides, such as deoxyhypoxanthine, inosine, 1-(2'-deoxy-β-D-rifuranosyl)-3-nitropyrrole, 5-nitroindole, or locked nucleic acids (LNAs).

[0214] In the method of this invention, the guide probe is not limited in length. For example, the guide probe can be 15-150 nt in length, such as 15-20 nt, 20-30 nt, 30-40 nt, 40-50 nt, 50-60 nt, 60-70 nt, 70-80 nt, 80-90 nt, 90-100 nt, 100-110 nt, 110-120 nt, 120-130 nt, 130-140 nt, or 140-150 nt. The first target-specific sequence in the guide probe can be of any length, as long as it can specifically hybridize with the target nucleic acid sequence. For example, the length of the first target-specific sequence in the guide probe can be 10-140 nt, such as 10-20 nt, 20-30 nt, 30-40 nt, 40-50 nt, 50-60 nt, 60-70 nt, 70-80 nt, 80-90 nt, 90-100 nt, 100-110 nt, 110-120 nt, 120-130 nt, 130-140 nt. In some preferred embodiments, the length of the first target-specific sequence in the guide probe is 10-100 nt (e.g., 10-90 nt, 10-80 nt, 10-50 nt, 10-40 nt, 10-30 nt, 10-20 nt).

[0215] In the method of the present invention, the guide probe can also be modified, for example, to make it resistant to nuclease activity (e.g., 5' nuclease activity, such as 5' to 3' exonuclease activity, 5' to 3' endonuclease activity). For example, modifications resistant to nuclease activity can be introduced into the backbone of the detection probe, such as thiophosphate bonds, alkyl phosphate triester bonds, aryl phosphate triester bonds, alkyl phosphonate bonds, aryl phosphonate bonds, hydrogenated phosphate bonds, alkyl amino phosphate bonds, aryl amino phosphate bonds, 2'-O-aminopropyl modification, 2'-O-alkyl modification, 2'-O-allyl modification, 2'-O-butyl modification, and 1-(2'-thio-PD-rifuranosyl) modification.

[0216] Upstream and downstream primers

[0217] As used herein, the term "upstream primer" refers to an oligonucleotide sequence containing a sequence complementary to the target nucleic acid sequence, which is capable of hybridizing (or annealing) with the target nucleic acid sequence under conditions allowing nucleic acid hybridization (or annealing) or amplification, and, when hybridizing with the target nucleic acid sequence, is located upstream of a first target-specific sequence or has at least partial overlap with the first target-specific sequence. In this application, the upstream primer is not limited to a specific sequence, as long as it contains a sequence complementary to the target nucleic acid and is capable of hybridizing with the target nucleic acid. As used herein, the term "downstream primer" refers to an oligonucleotide sequence containing a sequence complementary to the target nucleic acid sequence, which is capable of hybridizing (or annealing) with the target nucleic acid sequence under conditions allowing nucleic acid hybridization (or annealing) or amplification, and, when hybridizing with the target nucleic acid sequence, is located downstream of a second target-specific sequence or has at least partial overlap with the second target-specific sequence. In this application, the downstream primer is not limited to a specific sequence, as long as it contains a sequence complementary to the target nucleic acid and is capable of hybridizing with the target nucleic acid.

[0218] In some embodiments of the invention, the upstream primer may comprise or consist of naturally occurring nucleotides (e.g., deoxyribonucleotides or ribonucleotides), modified nucleotides, non-natural nucleotides, or any combination thereof. In some preferred embodiments, the upstream primer comprises or consists of natural nucleotides (e.g., deoxyribonucleotides or ribonucleotides). In some preferred embodiments, the upstream primer comprises modified nucleotides, such as modified deoxyribonucleotides or ribonucleotides, such as 5-methylcytosine or 5-hydroxymethylcytosine. In some preferred embodiments, the upstream primer comprises non-natural nucleotides, such as deoxyxanthine, inosine, 1-(2'-deoxy-β-D-rifuranosyl)-3-nitropyrrole, 5-nitroindole, or locked nucleic acid (LNA).

[0219] In the method of this invention, the upstream primer is not limited in length, as long as it can specifically hybridize with the target nucleic acid sequence. For example, the length of the upstream primer can be 15-150 nt, such as 15-20 nt, 20-30 nt, 30-40 nt, 40-50 nt, 50-60 nt, 60-70 nt, 70-80 nt, 80-90 nt, 90-100 nt, 100-110 nt, 110-120 nt, 120-130 nt, 130-140 nt, or 140-150 nt.

[0220] In some preferred embodiments, the upstream primer, after hybridization with the target nucleic acid, is located distally upstream of the guide probe. In some preferred embodiments, the upstream primer, after hybridization with the target nucleic acid, is located adjacent to the guide probe upstream of it. In some preferred embodiments, the upstream primer, after hybridization with the target nucleic acid, has a partially overlapping sequence with the guide probe (e.g., the 3' portion of the upstream primer partially overlaps with the 5' portion of the guide probe). In some preferred embodiments, the upstream primer, after hybridization with the target nucleic acid, has a completely overlapping sequence with the guide probe. In some preferred embodiments, the upstream primer, after hybridization with the target nucleic acid, completely contains the sequence of the guide probe.

[0221] In some preferred embodiments, the upstream primer is a primer or a probe specific to the target nucleic acid sequence.

[0222] In some embodiments of the invention, the downstream primer may comprise or consist of naturally occurring nucleotides (e.g., deoxyribonucleotides or ribonucleotides), modified nucleotides, non-natural nucleotides, or any combination thereof. In some preferred embodiments, the downstream primer comprises or consists of natural nucleotides (e.g., deoxyribonucleotides or ribonucleotides). In some preferred embodiments, the downstream primer comprises modified nucleotides, such as modified deoxyribonucleotides or ribonucleotides, such as 5-methylcytosine or 5-hydroxymethylcytosine. In some preferred embodiments, the downstream primer comprises non-natural nucleotides, such as deoxyhypoxanthine, inosine, 1-(2'-deoxy-β-D-rifuranosyl)-3-nitropyrrole, 5-nitroindole, or locked nucleic acid (LNA).

[0223] In the method of this invention, the downstream primer is not limited in length, as long as it can specifically hybridize with the target nucleic acid sequence. For example, the length of the downstream primer can be 15-150 nt, such as 15-20 nt, 20-30 nt, 30-40 nt, 40-50 nt, 50-60 nt, 60-70 nt, 70-80 nt, 80-90 nt, 90-100 nt, 100-110 nt, 110-120 nt, 120-130 nt, 130-140 nt, or 140-150 nt.

[0224] In some embodiments of the invention, the upstream and downstream primers serve as target-specific primer pairs for amplifying the target nucleic acid. Depending on the method for amplifying the target nucleic acid, one or more pairs of target-specific primers may be used for each target nucleic acid. In some preferred embodiments, the target nucleic acid sequence is amplified in a symmetrical manner. In such embodiments, equal amounts of upstream and downstream primers are used for a given target nucleic acid sequence. In some preferred embodiments, the target nucleic acid sequence is amplified in an asymmetric manner. In such embodiments, unequal amounts of upstream and downstream primers are used for a given target nucleic acid sequence. In some embodiments, the upstream primer is in excess relative to the downstream primer (e.g., at least 1-10 times excess). In some embodiments, the downstream primer is in excess relative to the upstream primer (e.g., at least 1-10 times excess).

[0225] Composition

[0226] On the other hand, this application provides a composition comprising: a polymerase having 5' nuclease activity, and at least one guide probe and at least one first oligonucleotide probe for each target nucleic acid to be detected; wherein,

[0227] The guide probe includes a first target-specific sequence comprising a sequence at least partially complementary to a first region of a target nucleic acid; optionally, the guide probe further includes a second sequence downstream of the first target-specific sequence; and

[0228] The first oligonucleotide probe includes a mediator sequence and a second target-specific sequence from the 5' to 3' direction, the mediator sequence comprising a sequence that is not complementary to the target nucleic acid; and the second target-specific sequence comprising a sequence that is at least partially complementary to a second region of the target nucleic acid;

[0229] The target nucleic acid comprises a first region and a second region, with the first region located downstream of the second region.

[0230] In some embodiments, the nucleotide at the 5' end of the second target-specific sequence is complementary to a second region of the target nucleic acid. In some embodiments, the regions where the guide probe and the first oligonucleotide probe hybridize with the target nucleic acid are close to each other (e.g., adjacent, adjacent to, or partially overlapping).

[0231] The guide probe does not contain a second sequence.

[0232] In some implementations, the guide probe does not contain a second sequence.

[0233] In some implementations, the first and second regions of the target nucleic acid are adjacent to each other.

[0234] In some embodiments, when hybridizing with the target nucleic acid, the first target-specific sequence hybridizes in region A of the target nucleic acid, and the second target-specific sequence hybridizes in region B of the target nucleic acid; and, in the target nucleic acid, region A is located downstream of region B and adjacent to region B.

[0235] In some embodiments, region A is separated from region B by 5, 4, 3, 2, 1, or 0 nucleotides. In some preferred embodiments, region A is separated from region B by 2, 1, or 0 nucleotides.

[0236] 3' end

[0237] In this document, the guide probe is not limited to the nucleotides of the 3' portion (e.g., the 3' end). Therefore, in some embodiments, when the guide probe does not contain a second sequence, the nucleotides of the 3' portion (e.g., the 3' end) of the first target-specific sequence may be complementary to or not complementary to the target nucleic acid. In some embodiments, when the guide probe does not contain a second sequence, the first target-specific sequence has a 3'-OH end, or its 3' end is closed. As used herein, the term "3'-terminal nucleotide" refers to the first nucleotide at the 3' end.

[0238] 3' end closed

[0239] In some implementations, the 3' end of the first target-specific sequence is closed.

[0240] In some embodiments, the 3' end of the guide probe is blocked by modifying the 3'-OH of the nucleotide at the 3' end of the first target-specific sequence (e.g., by modifying an amino group or a phosphate group); or by adding a chemical moiety (e.g., biotin, alkyl, or a quencher group) to the 3'-OH of the nucleotide at the 3' end of the first target-specific sequence; or by removing the 3'-OH of the nucleotide at the 3' end of the first target-specific sequence; or by replacing the nucleotide at the 3' end of the first target-specific sequence with a dideoxynucleotide to block the 3' end of the guide probe.

[0241] In some embodiments, when the guide probe does not contain a second sequence, the 3' end of the guide probe is blocked by modifying the 3'-OH of the nucleotide at the 3' end of the first target-specific sequence with an amino group.

[0242] In some implementations, when the guide probe does not contain a second sequence, region A is separated from region B by 0 nucleotides.

[0243] In some implementations, when the guide probe does not contain a second sequence, the nucleotide at the 3' end of the first target-specific sequence is complementary to the target nucleic acid.

[0244] In some embodiments, when the guide probe does not contain a second sequence, the nucleotide at the 3' end of the first target-specific sequence is complementary to the target nucleic acid, and the 3' end is closed (e.g., the nucleotide at the 3' end has an amino group modification); and the region A is separated from the region B by 0 nucleotides.

[0245] In some embodiments, when the guide probe does not contain a second sequence, the 3' end nucleotide of the first target-specific sequence is complementary to the target nucleic acid, and the 3' end is closed (e.g., the 3' end nucleotide has an amino group modification); and the region A is separated from the region B by 2, 1, or 0 nucleotides. In such embodiments, a polymerase derived from eubacteria is used.

[0246] 3' end not closed

[0247] In some embodiments, the first target-specific sequence has a 3'-OH terminus.

[0248] In some implementations, when the guide probe does not contain a second sequence, the nucleotide at the 3' end of the first target-specific sequence is not complementary to the target nucleic acid.

[0249] In some embodiments, when the guide probe does not contain a second sequence, one or more nucleotides of the 3' portion of the first target-specific sequence are not complementary to the target nucleic acid. In some embodiments, when the guide probe does not contain a second sequence, multiple consecutive nucleotides at the 3' end of the first target-specific sequence are not complementary to the target nucleic acid.

[0250] The guide probe contains a second sequence

[0251] In some implementations, the guide probe further includes a second sequence downstream of the first target-specific sequence.

[0252] In some implementations, the first and second regions of the target nucleic acid are adjacent to each other.

[0253] In some embodiments, when hybridizing with the target nucleic acid, the first target-specific sequence hybridizes to region A of the target nucleic acid, and the second target-specific sequence hybridizes to region B of the target nucleic acid; and, in the target nucleic acid, region A and region B are adjacent (e.g., separated by 0 nucleotides).

[0254] In some embodiments, when hybridizing with the target nucleic acid, the second sequence is adjacent to the first target-specific sequence, and the length of the second sequence is 5, 4, 3, 2, or 1 nucleotide. In some preferred embodiments, the length of the second sequence is 3, 2, or 1 nucleotide.

[0255] 3' end

[0256] In this document, the guide probe is not limited to a 3' portion (e.g., a 3' end). Therefore, in some embodiments, when the guide probe also contains a second sequence downstream of the first target-specific sequence, the nucleotides of the 3' portion (e.g., the 3' end) of the second sequence may be complementary or non-complementary to the target nucleic acid. In some embodiments, the second sequence has a 3'-OH end, or its 3' end is closed. As used herein, the term "3'-terminal nucleotide" refers to the first nucleotide at the 3' end.

[0257] 3' end closed

[0258] In some implementations, the 3' end of the second sequence is closed.

[0259] In some embodiments, the 3' end of the guide probe is blocked by modifying the 3'-OH of the nucleotide at the 3' end of the second sequence (e.g., by modifying an amino group or a phosphate group); or by adding a chemical moiety (e.g., biotin, alkyl, or a quencher group) to the 3'-OH of the nucleotide at the 3' end of the second sequence; or by removing the 3'-OH of the nucleotide at the 3' end of the second sequence; or by replacing the nucleotide at the 3' end of the second sequence with a dideoxynucleotide to block the 3' end of the guide probe.

[0260] In some embodiments, when the guide probe contains a second sequence, the 3' end of the guide probe is blocked by modifying the 3'-OH of the nucleotide at the 3' end of the second sequence with an amino group.

[0261] In some embodiments, when the guide probe hybridizes with the target nucleic acid, the second sequence is adjacent to the first target-specific sequence, and the length of the second sequence is 3, 2, or 1 nucleotide.

[0262] In some implementations, when the guide probe contains a second sequence, the nucleotide at the 3' end of the second sequence is complementary to the target nucleic acid.

[0263] In some embodiments, one or more nucleotides of the 3' portion of the second sequence are complementary to the target nucleic acid. In some embodiments, multiple consecutive nucleotides at the 3' end of the second sequence are complementary to the target nucleic acid.

[0264] 3' end not closed

[0265] In some embodiments, the second sequence has a 3'-OH terminus.

[0266] In some embodiments, when the guide probe hybridizes with the target nucleic acid, the second sequence is adjacent to the first target-specific sequence, and the length of the second sequence is 3, 2, or 1 nucleotide.

[0267] In some implementations, when the guide probe contains a second sequence, the nucleotide at the 3' end of the second sequence is not complementary to the target nucleic acid.

[0268] In some embodiments, one or more nucleotides of the 3' portion of the second sequence are not complementary to the target nucleic acid. In some embodiments, multiple consecutive nucleotides at the 3' end of the second sequence are not complementary to the target nucleic acid.

[0269] In some embodiments, when the guide probe includes a second sequence, the nucleotide at the 3' end of the second sequence is not complementary to the target nucleic acid, and the nucleotide has a 3'-OH end; and the region A is separated from the region B by 0 nucleotides, and the length of the second sequence is 1 nucleotide.

[0270] In some implementations, for each target nucleic acid to be detected, at least one upstream primer is also provided; wherein the upstream primer contains a sequence complementary to the target nucleic acid; and, when hybridizing with the target nucleic acid, the upstream primer is located upstream of the first target-specific sequence or has a sequence that at least partially overlaps with the first target-specific sequence.

[0271] In some embodiments, after hybridization with the target nucleic acid, the upstream primer is located distal upstream of the first target-specific sequence. In some embodiments, after hybridization with the target nucleic acid, the upstream primer is located adjacent upstream of the first target-specific sequence. In some embodiments, after hybridization with the target nucleic acid, the upstream primer has a partially overlapping sequence with the first target-specific sequence (e.g., the 3' portion of the upstream primer partially overlaps with the 5' portion of the first target-specific sequence). In some embodiments, after hybridization with the target nucleic acid, the upstream primer has a completely overlapping sequence with the first target-specific sequence. In some embodiments, after hybridization with the target nucleic acid, the upstream primer completely contains the first target-specific sequence.

[0272] In some embodiments, the composition is used to detect the presence of one or more target nucleic acids in a sample, or to characterize and / or quantify one or more target nucleic acids, or to distinguish multiple target nucleic acids, or to detect one or more single nucleotide polymorphism sites (SNPs) contained in target nucleic acids, or to identify SNP sites with different genotypes in multiple target nucleic acids.

[0273] It is readily understood that such compositions can be used to implement the methods of the present invention described in detail above. Therefore, the various technical features described in detail above regarding the upstream primer, guide probe, first oligonucleotide probe, and second oligonucleotide probe can also be applied to the upstream primer, guide probe, first oligonucleotide probe, and second oligonucleotide probe in the composition. Therefore, in some preferred embodiments, the guide probe is as defined above. In some preferred embodiments, the target nucleic acid and / or sample is as defined above. In some preferred embodiments, the polymerase having 5' nuclease activity is as defined above. In some preferred embodiments, the first oligonucleotide probe is as defined above.

[0274] In some embodiments, at least one second oligonucleotide probe is also provided, wherein at least a portion of the second oligonucleotide probe is complementary to all or part of the mediator sequence, and the second oligonucleotide probe is labeled with a detectable tag. In some preferred embodiments, the second oligonucleotide is as defined above. In some preferred embodiments, at least one second oligonucleotide probe is also provided, wherein at least a portion of the second oligonucleotide probe is complementary to all or part of the cleavage product sequence, and the second oligonucleotide probe is labeled with a detectable tag; wherein the cleavage product is as defined above.

[0275] In some implementations, the upstream primer is as defined above.

[0276] In some embodiments, the composition further comprises one or more components for amplifying the target nucleic acid (e.g., an enzyme, primers or primer pairs for amplifying the target nucleic acid), and contacts the one or more components with the target nucleic acid under conditions that allow nucleic acid amplification.

[0277] In some embodiments, the composition further comprises at least one downstream primer (e.g., a downstream primer as defined above).

[0278] Reagent test kit

[0279] On the other hand, this application provides a kit comprising the composition as described above.

[0280] It is readily understood that such kits can be used to implement the methods of the present invention described in detail above. Therefore, the various technical features described in detail above for target nucleic acid amplification can also be applied to the kits. Thus, in some embodiments, the kits further comprise one or more enzymes (e.g., enzymes with strand displacement activity (e.g., BST enzymes), polymerases).

[0281] In some embodiments, the kit further comprises: reagents for nucleic acid hybridization, reagents for cleavage of a first oligonucleotide probe, reagents for nucleic acid extension, reagents for nucleic acid amplification, or any combination thereof. Such reagents can be conventionally determined by those skilled in the art and include, but are not limited to, working buffers for enzymes (e.g., nucleic acid polymerases), dNTPs, water, and reagents containing ions (e.g., Mg²⁺). 2+ Solutions of single-stranded DNA-binding proteins (SSBs), or any combination thereof.

[0282] In some preferred embodiments, the compositions or kits of this application do not contain an enzyme having 5' nuclease activity but significantly reduced polymerization activity (e.g., activity reduced by 70%, 80%, or more than 90%), substantially absent, or completely absent. In some preferred embodiments, the compositions or kits of this application do not contain FEN1 enzyme (e.g., M0645S, NEB, USA).

[0283] Those skilled in the art can modify, substitute, or combine various technical features of the present invention based on the principles described in detail in this application, without departing from the spirit and scope of the invention. All such technical solutions and their variations are covered within the scope of the claims of this application or their equivalents.

[0284] Beneficial effects

[0285] Compared with existing technologies, the combination of the guide probe, the first oligonucleotide probe, and the polymerase with 5' nuclease activity in this application not only exhibits higher enzyme digestion specificity but also significantly improves the digestion rate. Furthermore, compared with existing technologies, the detection method using the composition of this application (guide probe, first oligonucleotide probe, and polymerase with 5' nuclease activity) achieves better detection results in both singleton and multiplex detection of target nucleic acids (simultaneous detection of multiple target nucleic acid sequences) (e.g., higher detection signal, shorter detection time, and the ability to detect longer amplicon sequences (e.g., from approximately 1000 bp to approximately 2500 bp)). Moreover, the nucleic acid detection method and composition of this application are adaptable to various nucleic acid amplification methods / platforms and can be applied in various detection scenarios (e.g., SNP site detection), showing broad application prospects. Attached Figure Description

[0286] Figure 1 schematically illustrates the positional relationship between the upstream primer, guide probe, and first oligonucleotide probe after hybridization with the target nucleic acid; where,

[0287] The upstream primer contains a sequence complementary to the target nucleic acid; and, when hybridizing with the target nucleic acid, the upstream primer is located upstream of the first target-specific sequence or has a sequence that at least partially overlaps with the first target-specific sequence; and

[0288] The guide probe includes a first target-specific sequence, the first target-specific sequence including a sequence that is at least partially complementary to a first region of the target nucleic acid; optionally, the guide probe further includes a second sequence downstream of the first target-specific sequence;

[0289] The first oligonucleotide probe includes a mediator sequence and a second target-specific sequence from the 5' to 3' direction. The mediator sequence includes a sequence that is not complementary to the target nucleic acid. The second target-specific sequence includes a sequence that is at least partially complementary to a second region of the target nucleic acid.

[0290] After hybridization with the target nucleic acid, the upstream primer, guide probe, and first oligonucleotide probe are arranged in parallel with each other in the same direction, with the upstream primer located upstream of the guide probe and the guide probe located upstream of the first oligonucleotide probe.

[0291] Furthermore, the target nucleic acid comprises a first region and a second region, with the first region located downstream of the second region;

[0292] exist Figure 1A In one embodiment, the guide probe does not contain a second sequence, and the regions where the guide probe and the first oligonucleotide probe hybridize with the target nucleic acid are adjacent to each other; in this embodiment, region A where the guide probe hybridizes with the target nucleic acid and region B where the first oligonucleotide probe hybridizes with the target nucleic acid are separated by one nucleotide.

[0293] exist Figure 1B In one embodiment, the guide probe does not contain a second sequence, and the regions where the guide probe and the first oligonucleotide probe hybridize with the target nucleic acid are adjacent to each other; in this embodiment, the region A where the guide probe hybridizes with the target nucleic acid and the region B where the first oligonucleotide probe hybridizes with the target nucleic acid are separated by 0 nucleotides.

[0294] exist Figure 1C In one embodiment, the guide probe comprises a second sequence, and the regions where the guide probe and the first oligonucleotide probe hybridize with the target nucleic acid partially overlap. In this embodiment, region A, where the guide probe hybridizes with the target nucleic acid, and region B, where the first oligonucleotide probe hybridizes with the target nucleic acid, overlap by one nucleotide.

[0295] Figure 2 shows three specific implementation schemes for detecting target nucleic acids;

[0296] exist Figure 2A In the implementation scheme (LAMP), two upstream primers (upstream primers F3 and F2), two downstream primers (downstream primers B3 and B2), a guide probe, a first oligonucleotide probe, and a second oligonucleotide probe are designed and provided for the target nucleic acid molecule; wherein, the second oligonucleotide probe is labeled with a fluorescent group and a quencher group; and, the second oligonucleotide probe emits a signal after being cleaved by polymerase;

[0297] exist Figure 2B In the implementation scheme (RPA), for the target nucleic acid molecule, two upstream primers (upstream primers 4S and 1S), three downstream primers (downstream primers 3a, 2a, and 5a), as well as a guide probe, a first oligonucleotide probe, and a second oligonucleotide probe are designed and provided; wherein, the second oligonucleotide probe is labeled with a fluorescent group and a quencher group; and, the second oligonucleotide probe emits a signal after being cleaved by polymerase;

[0298] exist Figure 2C In the implementation scheme (CPA), for the target nucleic acid molecule, an upstream primer (upstream primer F), a downstream primer (downstream primer R), a guide probe, a first oligonucleotide probe, and a second oligonucleotide probe are designed and provided; wherein, the second oligonucleotide probe is labeled with a fluorescent group and a quencher group; and, the second oligonucleotide probe emits a signal after being cleaved by polymerase;

[0299] In the detection process of the three implementation schemes described above, the upstream primer, downstream primer, guide probe, first oligonucleotide probe, and second oligonucleotide probe hybridize (anneal) with the target nucleic acid molecule, respectively. All upstream and downstream primers are extended by a polymerase with 5' nuclease activity. Simultaneously, the hybridization of the guide probe with the target nucleic acid leads to the corresponding first oligonucleotide probe being cleaved by a polymerase with 5' nuclease activity, thereby releasing the cleavage product. Subsequently, the cleavage product hybridizes to the second oligonucleotide probe, causing the second oligonucleotide probe to be cleaved by a polymerase with 5' nuclease activity, thereby releasing a fluorescent group. Thus, the presence of the target nucleic acid can be determined by detecting the signal emitted by the fluorescent group.

[0300] Figure 3A specific implementation scheme for detecting a target nucleic acid is shown; for the target nucleic acid molecule, an upstream primer, a downstream primer, a guide probe, a first oligonucleotide probe, and a second oligonucleotide probe are designed and provided; wherein, the second oligonucleotide probe includes, from the 3' to 5' direction, a template sequence and a capture sequence, the template sequence being complementary to the cleavage product of the first oligonucleotide probe or a portion thereof; wherein, the second oligonucleotide probe is labeled with a fluorescent group and a quencher group; and, the second oligonucleotide probe emits a signal upon hybridization with its complementary sequence;

[0301] During the detection process, the upstream primer, downstream primer, guide probe, first oligonucleotide probe, and second oligonucleotide probe hybridize (anneal) with the target nucleic acid molecule. The upstream and downstream primers are extended by a polymerase with 5' nuclease activity. Simultaneously, the hybridization of the guide probe with the target nucleic acid leads to the cleavage of the corresponding first oligonucleotide probe by the polymerase with 5' nuclease activity, releasing the cleavage product. Further, the polymerase uses the template sequence as a template to extend the cleavage product hybridized to the template sequence, causing the second oligonucleotide probe to be cleaved by the polymerase with 5' nuclease activity, releasing a fluorescent group. Therefore, the presence of the target nucleic acid can be determined by detecting the signal emitted by the fluorescent group.

[0302] Figure 4 A specific implementation scheme for detecting a target nucleic acid is shown; for the target nucleic acid molecule, an upstream primer, a downstream primer, a guide probe, a first oligonucleotide probe, and a second oligonucleotide probe are designed and provided; wherein, the second oligonucleotide probe includes, from the 3' to 5' direction, a template sequence and a capture sequence, the capture sequence being complementary to the cleavage product of the first oligonucleotide probe or a portion thereof; wherein, the second oligonucleotide probe is labeled with a fluorescent group and a quencher group; and, the second oligonucleotide probe emits a signal upon hybridization with its complementary sequence;

[0303] During the detection process, the upstream primer, downstream primer, guide probe, first oligonucleotide probe, and second oligonucleotide probe hybridize (anneal) with the target nucleic acid molecule. The upstream and downstream primers are extended by a polymerase with 5' nuclease activity. Simultaneously, the hybridization of the guide probe with the target nucleic acid leads to the cleavage of the corresponding first oligonucleotide probe by the polymerase with 5' nuclease activity, releasing the cleavage product. Further, the polymerase uses the capture sequence as a template to extend the cleavage product hybridized to the capture sequence, thereby forming a double strand. Thus, the presence of the target nucleic acid can be determined by melting curve analysis.

[0304] Figure 5A schematic diagram of the experimental design for Example 1 is shown.

[0305] Figure 6 The fluorescence differences before and after adding the guide probe (3'-OH) are shown in detection systems using different enzymes.

[0306] Figure 7 The results show the comparison of enzyme digestion activities of different enzymes in the detection system using different enzymes.

[0307] Figure 8 It shows different factors (dNTPs, thermal start, Mg) 2+ The effects of concentration and temperature on the digestion activity of Taq DNA polymerase.

[0308] Figure 9A This is a schematic diagram of the principle of Example 4. Figure 9B This is a fluorescence curve of the CPA amplification product detected by Taq DNA polymerase cleavage activity in Example 4.

[0309] Figure 10 The fluorescence curve of the CPA-Taq system for real-time detection of SARS-CoV-2-N template is shown.

[0310] Figure 11 The fluorescence curve of LAMP amplification products as detected by Taq DNA polymerase cleavage activity is shown.

[0311] Figure 12 The fluorescence curve of the LAMP-Taq system for real-time detection of SARS-CoV-2-N template is shown.

[0312] Figure 13 The fluorescence curve of RPA amplification products as detected by Taq DNA polymerase digestion activity is shown.

[0313] Figure 14 The effect of base matching at the (3'-OH)3' end of the guide probe on the digestion activity of Taq DNA polymerase was shown.

[0314] Figure 15 A schematic diagram showing the number of overlaps at the ends of the guide probe (3'-OH)3' described in the embodiments is shown. The cases of overlaps of -2, -1, 0, 1, 2, and 3 are shown sequentially.

[0315] Figure 16 The effect of the number of overlapping bases at the (3'-OH)3' end of the guide probe on the digestion activity of Taq DNA polymerase was shown.

[0316] Figure 17 The effects of different 3' end modifications of the guide probe (3'-block) on Taq DNA polymerase digestion activity were shown.

[0317] Figure 18 The effect of base matching at the 3' end of the guide probe (3'-block) on Taq DNA polymerase digestion activity was shown.

[0318] Figure 19 The effect of the number of overlapping bases at the 3' end of the guide probe (3'-block) on the digestion activity of Taq DNA polymerase was shown.

[0319] Figure 20 The effects of adding a guide probe (3'-blocking) and a guide probe (3'-OH) in the LAMP-Taq real-time detection system are compared.

[0320] Figure 21 A schematic diagram illustrating the principle of distinguishing SNP mutations in Example 15 is shown.

[0321] Figure 22 The fluorescence curves showing the distinguishing mutations using the guide probe (3'-block) in the LAMP-Taq real-time detection system are displayed.

[0322] Figure 23 The fluorescence curves that distinguish mutations in the LAMP-Taq real-time detection system are shown.

[0323] Figure 24 The mass spectrometry results of Taq DNA polymerase cutting wild / mutant templates under the action of the guide probe (3'-block) are shown.

[0324] Figure 25 The mass spectrometry results of Taq DNA polymerase cutting wild / mutant templates under the action of the guide probe (3'-OH) are shown.

[0325] Figure 26 The effect of adding a guide probe (3'-block) to the CPA-fluorescent probe detection system is shown.

[0326] Figure 27 The effect of adding a guide probe (3'-block) to the LAMP-fluorescent probe detection system is shown.

[0327] Figure 28 The results show a comparison of signal intensity in Mediator Probe PCR under the action of the guide probe (3'-OH).

[0328] Figure 29 The signal intensity of Mediator Probe PCR under the action of the guide probe (3'-block) is shown.

[0329] Figure 30The comparison results show the introduction of a guide probe (3'-OH) in the MeltArray single detection system.

[0330] Figure 31 The melting signal peaks before and after the introduction of the guide probe (3'-OH) in the MeltArray multiplex detection system are shown.

[0331] Figure 32 The effect of the number of targets on the signal intensity in the MeltArray multiplex detection system under the action of the guide probe (3'-OH) is shown.

[0332] Figure 33 The signal intensity of MeltArray under different base overlap numbers and the action of a guide probe modified at the 3' end (3'-blocking) is shown.

[0333] Figure 34 The concentration of the guide probe (3'-block) in the MeltArray single detection system is shown.

[0334] Figure 35 The effect of the number of targets on the signal intensity of the MeltArray multiplex detection system under the action of the guide probe (3'-closed) is shown.

[0335] Figure 36 The melting signal peaks before and after the introduction of the guide probe (3'-closed) in the MeltArray multiplex detection system are shown.

[0336] Figure 37 The sensitivity of the O5 gene introduction guide probe (3'-block) in single-detection assays is shown.

[0337] Figure 38 The sensitivity of the O5 gene introduction guide probe (3'-block) in multiplex detection is shown.

[0338] Figure 39 This study demonstrates the reduction of media subsequence extension duration in MeltArray multiple detection.

[0339] Figure 40 This study demonstrates the duration of the shortened mediator sequence extension in the O8 gene.

[0340] Figure 41 The changes in melting peak intensity are shown as the length of the amplified product increases.

[0341] Figure 42 The melting signal peaks before and after the addition of the guide probe (3'-block) are shown in a detection system in which the upstream primer and the guide probe (3'-block) partially overlap.

[0342] Figure 43The melting signal peaks before and after the addition of the guide probe (3'-block) are shown in a detection system in which the upstream primer and the guide probe (3'-block) are completely overlapped.

[0343] Figure 44 The fluorescence signal intensity was shown in the detection system containing FEN1 with no guide probe, with guide probe (3'-block), and with guide probe (3'-OH). Detailed Implementation

[0344] The present invention will now be described with reference to the following embodiments, which are intended to illustrate the invention (and not limit it). It should be understood that these embodiments are merely for illustrating the principles and technical effects of the invention, and do not represent all possibilities of the invention. The invention is not limited to the materials, reaction conditions, or parameters mentioned in these embodiments. Those skilled in the art can implement other technical solutions using other similar materials or reaction conditions based on the principles of the invention. Such technical solutions do not depart from the basic principles and concepts described in the invention and are covered within the scope of the invention.

[0345] Example 1. Verification of the cleavage activity of Taq DNA polymerase

[0346] The embodiments described herein involve two types of guide probes: guide probes with a 3'-closed base and guide probes with an unclosed 3'-terminal base. The guide probe with a 3'-closed base is referred to below as "guide probe (3'-closed)", and the guide probe with an unclosed 3'-terminal base is referred to below as "guide probe (3'-OH)".

[0347] The experimental design of this embodiment is as follows: Figure 5 As shown, the 5' end of the fluorescent probe and the middle of the probe are each labeled with a fluorescent group FAM and a quenching group BHQ1, respectively. The 3' end of the guide probe (3'-OH) forms a single-base overlapping structure with the detection site and does not match the template.

[0348] The experiment consisted of two reactions. Reaction 1 was composed of the following components: 0.2 μM Probe_1, 0.5 μM guide probe (3'-OH)_1, 1×thermol buffer (20 mM Tris-HCl, 10 mM (NH4)2SO4, 150 mM KCl, 2 mM MgSO4, 0.1%... 20 (pH 8.8 at 25℃), 6.0 mM MgCl2 and 1.0 U Taq DNA polymerase, with sterile water added to a final volume of 10 μL. Reaction 2 had the same components as reaction 1 but without the guide probe (3'-OH)_1. The reactions were performed on a SLAN-96S fluorescent PCR instrument (Shanghai Hongshi Medical Technology Co., Ltd., China), set at 65℃ for 50 minutes, with fluorescence detected simultaneously. The oligonucleotides used in the reactions were synthesized by Shanghai Bioengineering Co., Ltd. (Table 1).

[0349] The validation targets included different DNA polymerases (Zhishan Biotechnology Co., Ltd., China), FEN1 (Catalog#M0645S, NEB, USA), and Taq Exo (laboratory expression and purification). For details, please refer to FrontMicrobiol 2014, 3:5:461; Gene 1992, 112(1):29-35; PCR Methods Appl 1993, 2(4):275-87; Angew Chem Int Ed Engl 2010, 49(34):5921-4. Among them, Taq Exo only has the exonuclease domain of the complete Taq DNA polymerase, Klen-Taq only has the polymerase domain of the complete Taq DNA polymerase, and the other Taq DNA polymerases are all variants of the complete Taq DNA polymerase.

[0350] Table 1: Sequences Used

[0351]

[0352] Experimental results:

[0353] (1) In the reaction with the addition of guide probe (3'-OH), only the Klen-Taq DNA polymerase reaction did not produce fluorescence, while the other reactions all produced fluorescence signals.

[0354] (2) In the reactions without the guide probe (3'-OH), only the Taq Exo reaction produced a fluorescence signal, but this was significantly lower than in the reactions with the guide probe (3'-OH). No fluorescence was observed in the other reactions. Figure 6 ).

[0355] Experimental conclusions: After the addition of the guide probe (3'-OH), all polymerases with 5' nuclease activity showed significantly higher cleavage activity, while Klen-Taq, which lacks 5' nuclease activity, consistently showed no cleavage activity. Furthermore, based on the activity comparison of Taq Exo with other enzymes, after the addition of the guide probe (3'-OH), the cleavage activity of enzymes possessing both 5' nuclease and polymerization activities was essentially the same as that of enzymes with only 5' nuclease activity.

[0356] Example 2. Comparison of enzymatic digestion activities of Taq DNA polymerase and FEN1 enzyme

[0357] The experimental design in this embodiment is the same as in "Example 1". Referring to the FEN1 enzyme (M0645S, NEB, USA) manual, we define the activity unit of Taq DNA polymerase as the enzyme capable of cleaving 1 μmol / L of substrate in a 10 μL reaction system within 10 min at 63°C, i.e., 1U = 0.1 μmol / (L·min). Simulating an enzyme excess scenario, the reaction was carried out for a sufficiently long time to completely consume the substrate. A linear relationship between the fluorescence increase and the amount of substrate consumed was obtained, and the enzyme activities of Taq DNA polymerase and FEN1 enzyme were calculated.

[0358] The specific components of the reaction system are as follows: 0.4 μM Probe_1, 1 μM guiding probe (3'-OH)_1, 1×thermol buffer (20 mM Tris-HCl, 10 mM (NH4)2SO4, 150 mM KCl, 2 mM MgSO4, 0.1%... 20 μL (pH 8.8 at 25℃), 2.0 mM MgCl2, and 8.0 U Taq DNA polymerase / 32.0 U FEN1 enzyme were added, and sterile water was added to a final volume of 10 μL. The reaction was performed on a SLAN-96S fluorescent PCR instrument (Shanghai Hongshi Medical Technology Co., Ltd., China) at 65℃ for 50 minutes, with fluorescence detected simultaneously. The oligonucleotides used were the same as those in Table 1.

[0359] Experimental results: The digestion activities of different Taq DNA polymerases were 8 to 20 times that of FEN1 enzyme. Figure 7 ).

[0360] Experimental conclusion: Taq DNA polymerase per unit volume exhibited stronger enzymatic digestion activity than FEN1 enzyme per unit volume, indicating that Taq DNA polymerase has a greater advantage in enzymatic digestion activity.

[0361] Example 3. Effect of reaction conditions on Taq DNA polymerase digestion activity

[0362] Experimental objective: To investigate the effects of temperature and Mg 2+ The effects of four factors—concentration, hot start, and dNTPs—on the digestion activity of Taq DNA polymerase.

[0363] Experimental design: When investigating the effect of temperature, the temperature range is between 55℃ and 80℃. The study investigates the effect of Mg... 2+ When considering the effect of concentration, the concentration range was between 0 mM and 10 mM. To investigate the effect of hot start, control groups were set up with and without a hot start step. When investigating the effect of dNTPs, control groups were set up with and without added dNTPs. Besides temperature and Mg... 2+ Apart from the four factors of concentration, hot start and dNTPs, the other reaction components and reaction conditions are the same as in "Example 1", and the oligonucleotides used are the same as those in Table 1.

[0364] Experimental results ( Figure 8 ):

[0365] 1. dNTPs have little effect on the digestion activity of Taq DNA polymerase; the addition or absence of dNTPs does not significantly change the digestion activity.

[0366] 2. Hot start has little effect on the digestion activity of Taq DNA polymerase; setting a hot start step or not will not significantly change the digestion activity.

[0367] 3. Temperature has a certain influence on the digestion activity of Taq DNA polymerase, with the optimal temperature being between 70 and 80℃.

[0368] 4. Mg 2+ The concentration of Mg has a certain influence on the digestion activity of Taq DNA polymerase. 2+ The optimal concentration is between 4 and 6 mM.

[0369] Experimental conclusion: For Taq DNA polymerase, hot start and dNTPs have little effect on enzyme digestion activity. Temperature and Mg 2+ Concentration can affect enzyme digestion activity to some extent. The most suitable reaction conditions are a temperature above 50℃ and the presence of Mg in the reaction. 2+ .

[0370] Example 4. Detection of CPA amplification products—Taq DNA polymerase-based guide probe (3'-OH) Assay

[0371] Objective: To investigate the feasibility of using a Taq DNA polymerase-based guide probe (3'-OH) assay to detect CPA reaction products.

[0372] Experimental Design: The experimental principle is the same as in "Example 3", and the schematic diagram is as follows. Figure 9AAs shown. The SARS-CoV-2N gene was selected as the target gene in the experiment. The nucleotide sequence of the target gene was downloaded from GenBank, and its specificity was determined using NCBI BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). Primer Premier5, TMUtility1.5, and T... m Online prediction software (Integrated DNA Technologies Biophysic, https: / / biophysics.idtdna.com / ) can be used to assist in the design of primer, probe, and guide probe (3'-OH) sequences.

[0373] The first step of the experiment was to establish the CPA reaction system. Using the SARS-Cov-2-N gene as the target, cross-primer sequences CPA_N_1s, CPA_N_2a, CPA-N-3a, CPA_N_4s, and CPA_N_5a were designed, synthesized, and screened to establish the corresponding CPA reaction system. The second step was to optimize the reaction conditions, including the concentration of Bst enzyme, reaction temperature, and time. After optimization, standard nucleic acid samples and positive controls were prepared for reaction verification and performance evaluation. The third step involved designing, synthesizing, and screening the corresponding guide probe (3'-OH)_2, first oligonucleotide probe_1, and second oligonucleotide probe_1 based on the established CPA system.

[0374] The final reaction components are as follows: 0.8 μM CPA_N_1s, 0.4 μM CPA_N_2a / 3a, 0.5 μM CPA_N_4s, 0.2 μM CPA_N_5a, 1×thermol buffer (20 mM Tris-HCl, 10 mM (NH4)2SO4, 150 mM KCl, 2 mM MgSO4, 0.1%) 20 (pH 8.8 at 25℃), 6.0 mM MgCl2, 8.0 U BST enzyme and 5 μL template DNA, and sterile water to a final volume of 25 μL.

[0375] After completing the above steps, the CPA reaction product was detected using a Taq DNA polymerase-based guide probe (3'-OH)Assay. This reaction consisted of two steps: the first step was the CPA reaction, performed on a SLAN-96S fluorescent PCR instrument (Shanghai Hongshi Medical Technology Co., Ltd., China) at 65℃ for 50 minutes; the second step was the detection reaction, in which the product was opened, and 0.2 μM guide probe (3'-OH)_2, 0.4 μM first oligonucleotide probe_1, 0.4 μM second oligonucleotide probe_1, and 8.0 U Taq DNA polymerase were added to the CPA amplicon. This was then performed on a SLAN-96S fluorescent PCR instrument (Shanghai Hongshi Medical Technology Co., Ltd., China) at 65℃ for 50 minutes, with fluorescence detected simultaneously. The oligonucleotides used in the reaction were synthesized by Shanghai Bioengineering Co., Ltd. (Table 2).

[0376] Table 2: Sequences Used

[0377]

[0378] Experimental results: The reaction with CPA amplification products produced a fluorescent signal, while no fluorescent signal was observed in the negative control. Figure 9B ).

[0379] Experimental conclusion: The CPA reaction product can be detected using a Taq DNA polymerase-based guide probe (3'-OH)Assay.

[0380] Example 5. Establishment of CPA-Taq Real-time Detection System

[0381] Experimental objective: To investigate the feasibility of Taq DNA polymerase-based guide probe (3'-OH)Assay cascade and to establish a CPA-Taq real-time detection system.

[0382] Experimental Design: The CPA-SARS-CoV-2-N system used in this experiment is the same as in "Example 4". Based on the established CPA system components, Taq DNA polymerase and the components required for the guide probe (3'-OH)Assay were added to ensure that the CPA reaction and the guide probe (3'-OH)Assay occur simultaneously.

[0383] The specific components of the reaction are as follows: 0.8 μM CPA_N_1s, 0.4 μM CPA_N_2a / 3a, 0.5 μM CPA_N_4s, 0.2 μM CPA_N_5a, 0.2 μM second oligonucleotide probe_1, 0.5 μM guide probe (3'-OH)_2, 0.4 μM first oligonucleotide probe_1, 0.5 μM 1×thermol buffer (20 mM Tris-HCl, 10 mM (NH4)2SO4, 150 mM KCl, 2 mM MgSO4, 0.1%) The reaction mixture consisted of 20 μL (pH 8.8 at 25℃), 6.0 mM MgCl2, 8.0 U BST Polymerase, 8.0 U Taq DNA polymerase, and 5 μL template DNA, with sterile water added to a final volume of 25 μL. The reaction was performed on a SLAN-96S fluorescent PCR instrument (Shanghai Hongshi Medical Technology Co., Ltd., China) at 65℃ for 50 minutes, with fluorescence detected simultaneously. The oligonucleotides used in the reaction were synthesized by Shanghai Bioengineering Co., Ltd. (Table 3).

[0384] Table 3: Sequences Used

[0385]

[0386] Experimental results: The positive reaction produced a fluorescent signal, while no fluorescence was observed in the negative control. Figure 10 ).

[0387] Experimental conclusion: The application of the Taq DNA polymerase-based guide probe (3'-OH)Assay in real-time CPA detection was successfully realized. The detection process does not involve opening the lid, minimizing the risk of contamination, and is simple to operate. Furthermore, the CPA-Taq system exhibits short detection time and high fluorescence signal.

[0388] Example 6. Detection of LAMP amplification products – using a Taq DNA polymerase-guided probe (3'-OH) Assay

[0389] Experimental objective: To investigate the feasibility of using a Taq DNA polymerase-based guide probe (3'-OH)Assay to detect LAMP reaction products.

[0390] Experimental Design: The first step of this experiment was the establishment of the LAMP reaction system. Using the SARS-Cov-2-N gene as the target, primer sequences LAMP_N_F3, LAMP_N_B3, LAMP_N_FIP, LAMP_N_BIP, LAMP_N_LF, and LAMP_N_LB were designed, synthesized, and screened to establish the corresponding LAMP system. The second step was to optimize the reaction conditions, including the concentration of Bst enzyme, reaction temperature, and time. After optimization, standard nucleic acid samples and positive controls were prepared for reaction verification and performance evaluation. The third step was to design, synthesize, and screen the corresponding guide probe (3'-OH)_2, first oligonucleotide probe_1, and second oligonucleotide probe_1 based on the established LAMP system.

[0391] The final reaction components are as follows: 0.2 μM LAMP_N_F3 / B3, 2 μM LAMP_N_FIP / BIP, 0.4 μM LAMP_N_LF / BF, 1×thermol buffer (20 mM Tris-HCl, 10 mM (NH4)2SO4, 150 mM KCl, 2 mM MgSO4, 0.1%) 20 (pH 8.8 at 25℃), 2mM MgCl2, 1mM dNTPs, 8.0U BST Polymerase and 5μL template DNA, then add sterile water to a final volume of 25μL.

[0392] After completing the above steps, the LAMP reaction product was detected using a Taq DNA polymerase-based guide probe (3'-OH)Assay. This reaction consisted of two steps: the first step was the LAMP reaction, performed on a SLAN-96S fluorescent PCR instrument (Shanghai Hongshi Medical Technology Co., Ltd., China) at 65℃ for 50 minutes; the second step was the detection reaction, in which the product was opened, and 0.2 μM guide probe (3'-OH)_2, 0.4 μM first oligonucleotide probe_1, 0.2 μM second oligonucleotide probe_1, and 8.0 U Taq DNA polymerase were added to the LAMP amplicon. This was then performed on a SLAN-96S fluorescent PCR instrument (Shanghai Hongshi Medical Technology Co., Ltd., China) at 65℃ for 50 minutes, with fluorescence detected simultaneously. The oligonucleotides used in the reaction were synthesized by Shanghai Bioengineering Co., Ltd. (Table 4).

[0393] Table 4: Sequences Used

[0394]

[0395] Experimental results: The reaction containing LAMP amplification products produced a fluorescent signal, while no fluorescent signal was observed in the negative control. Figure 11).

[0396] Experimental conclusion: The LAMP reaction products can be detected using the Taq DNA polymerase-based guide probe (3'-OH)Assay.

[0397] Example 7. Establishment of a LAMP-Taq real-time detection system

[0398] Experimental objective: To verify the feasibility of cascading the LAMP reaction with a Taq DNA polymerase-based guide probe (3'-OH)Assay and to establish a real-time LAMP-Taq detection system.

[0399] Experimental Design: The LAMP-SARS-CoV-2-N system used in this experiment was the same as in "Example 5". Thermostable Taq DNA polymerase and the components required for the guide probe (3'-OH)Assay were added to the established LAMP system components, allowing the LAMP reaction and the guide probe (3'-OH)Assay to proceed simultaneously.

[0400] The final reaction components are as follows: 0.6 μM first oligonucleotide probe_1, 0.2 μM second oligonucleotide probe_1, 0.2 μM LAMP_N_F3 / B3, 2 μM LAMP_N_FIP / BIP, 0.4 μM LAMP_N_LF / BF, 0.5 μM guide probe (3'-OH)_2, 1×thermol buffer (20 mM Tris-HCl, 10 mM (NH4)2SO4, 150 mM KCl, 2 mM MgSO4, 0.1%) The following reagents were added: 20 μL template DNA (pH 8.8 at 25℃), 2 mM MgCl2, 1 mM dNTPs, 8.0 U BST Polymerase and 8.0 U Taq DNA polymerase, and 5 μL template DNA. Sterile water was added to a final volume of 25 μL. The reaction was performed on a SLAN-96S fluorescent PCR instrument (Shanghai Hongshi Medical Technology Co., Ltd., China) at 65℃ for 50 minutes, with fluorescence detected simultaneously. The oligonucleotides used in the reaction were synthesized by Shanghai Bioengineering Co., Ltd. (Table 5).

[0401] Table 5: Sequences Used

[0402]

[0403]

[0404] Experimental results: The reaction with added template DNA produced a fluorescent signal, while no fluorescent signal was observed in the negative control. Figure 12 ).

[0405] Experimental conclusions: The application of the Taq DNA polymerase-based guide probe (3'-OH) assay in real-time LAMP detection was successfully realized. The detection process does not involve opening the tube cap, minimizing the risk of contamination; the operation is simple. The LAMP-Taq system exhibits short detection time, high detection signal, and high detection sensitivity.

[0406] Example 8. Detection of RPA-Amplification Products – A Guide Probe Based on Taq DNA Polymerase (3'-OH) Assay

[0407] Experimental objective: To investigate the feasibility of using a Taq DNA polymerase-based guide probe (3'-OH)Assay to detect RPA reaction products.

[0408] Experimental Design: The first step of this experiment was to establish the RPA reaction system. Using the SARS-Cov-2-N gene as the target, primer sequences RPA_N_F1 and RPA_N_R1 were designed, synthesized, and screened to establish the corresponding RPA reaction system. The second step was to optimize the reaction conditions, including the concentration of Bst enzyme, reaction temperature, and time. After optimization, standard nucleic acid samples and positive controls were prepared for reaction verification and performance evaluation. The third step was to design, synthesize, and screen the corresponding guide probe (3'-OH)_2, first oligonucleotide probe_1, and second oligonucleotide probe_1 based on the established RPA system sequence.

[0409] The RPA reaction was performed using the DNA Isothermal Rapid Amplification Kit (Basic Type) (AMP Future, China). The reaction components were as follows: 1×Reaction Buffer A, 1×Reaction Buffer B, 0.4 μM RPA_N_F1 and RPA_N_R1, 4 μL template, and sterile water to a final volume of 25 μL.

[0410] After completing the above steps, the RPA reaction product was detected using a Taq DNA polymerase-based guide probe (3'-OH)Assay. This reaction consisted of two steps: the first step was the RPA reaction, performed on a SLAN-96S fluorescent PCR instrument (Shanghai Hongshi Medical Technology Co., Ltd., China) at 65℃ for 30 minutes; the second step was the detection reaction, in which the product was opened, and 0.2 μM guide probe (3'-OH)_2, 0.4 μM first oligonucleotide probe_1, 0.4 μM second oligonucleotide probe_1, and 8.0 U Taq DNA polymerase were added to the RPA amplification product. This was then performed on a SLAN-96S fluorescent PCR instrument (Shanghai Hongshi Medical Technology Co., Ltd., China) at 65℃ for 50 minutes, with fluorescence detected simultaneously. The oligonucleotides used in the reaction were synthesized by Shanghai Bioengineering Co., Ltd. (Table 6).

[0411] Table 6: Sequences Used

[0412]

[0413] Experimental results: The reaction containing RPA amplification products produced a fluorescent signal, while no fluorescent signal was observed in the negative control. Figure 13 ).

[0414] Experimental conclusion: The RPA reaction products can be detected using the Taq DNA polymerase-based guide probe (3'-OH)Assay.

[0415] Example 9. Effect of base difference at the 3' end of the guide probe (3'-OH) on Taq DNA polymerase digestion activity

[0416] Objective: To investigate the effect of whether the 3' end base of the guide probe (3'-OH) matches the template on the digestion activity of Taq DNA polymerase.

[0417] Experimental Design: The LAMP-Taq real-time detection system used in the experiment was the same as in "Example 6". The guide probes (3'-OH) used in the experiment had A, T, C, and G bases at their 3' ends, with the guide probes (3'-OH) having a C base at their 3' end matching the template.

[0418] The reaction components and reaction conditions are the same as in "Example 6". The oligonucleotides used are shown in Table 7.

[0419] Table 7. Sequences Used

[0420]

[0421]

[0422] Experimental results: The fluorescence signal of the reaction with the addition of a guide probe (3'-OH) whose 3' terminal base does not match the template was significantly higher than that of the reaction with the addition of a guide probe (3'-OH) whose 3' terminal base matches the template. Furthermore, the reaction signal with the addition of a guide probe (3'-OH) whose 3' terminal base matches the template was similar to that without the addition of a guide probe (3'-OH). Figure 14 ).

[0423] Experimental conclusion: Whether the 3' end base of the guide probe (3'-OH) matches the template has a significant impact on the activity of Taq DNA polymerase.

[0424] Example 10. Effect of the number of overlapping bases at the 3' end of the guide probe (3'-OH) on the digestion activity of Taq DNA polymerase.

[0425] Objective: To verify the effect of the number of overlapping bases at the 3' end of the guide probe (3'-OH) on the digestion activity of Taq DNA polymerase.

[0426] Experimental Design: The experiment used the LAMP-Taq real-time detection system as in "Example 6". The definition of the number of overlapping bases at the 3' end is as follows: Figure 15 The experiment used a guide probe (3'-OH) with an overlap of 2 to 3 bases at the 3' end, and the 3' ends were all natural bases that did not match the template.

[0427] The reaction components and reaction conditions are the same as in "Example 6". The oligonucleotides used are shown in Table 8.

[0428] Table 8. Sequences Used

[0429]

[0430]

[0431] Experimental results: The number of overlapping bases at the 3' end of the guide probe (3'-OH) has a certain influence on the enzymatic activity of Taq DNA polymerase. The reaction with guide probe (3'-OH) having 1 to 3 overlapping bases at the 3' end produces a fluorescent signal, while the reaction with guide probe (3'-OH) having 2 overlapping bases at the 3' end produces almost no fluorescent signal. The highest fluorescent signal is produced when the guide probe (3'-OH) has 1 overlapping base at the 3' end. Figure 16 ).

[0432] Example 11. Effect of 3' end modification of guide probe on Taq DNA polymerase digestion activity

[0433] Objective: To investigate the effect of 3' end modification of the guide probe on Taq DNA polymerase digestion activity.

[0434] Experimental Design: The LAMP-Taq real-time detection system used in the experiment was the same as in "Example 6". The guide probe used in the experiment had a natural base at the 3' end that did not match the template and different terminal modification groups, including amino groups, phosphate groups, spacers, biotin, dideoxynucleotides (ddT), and quenchers (BHQ1). The 3' terminal base of the former overlapped with the downstream duplex by 1 base, while the 3' terminal base of the latter overlapped with the downstream duplex by 0 bases.

[0435] The reaction components and reaction conditions are the same as in "Example 6". The oligonucleotides used are shown in Table 9.

[0436] Table 9. Sequences Used

[0437]

[0438]

[0439] Experimental results: Regarding fluorescence signal, the highest fluorescence signal was observed when the 3' end of the guide probe was a mismatched natural base, i.e., guide probe (3'-OH)_1_OH. The second highest fluorescence signal was observed when the 3' end of the guide probe (3'-blocked) was amino-modified. The fluorescence signal of guide probe (3'-blocked) with other groups added to its 3' end was lower than both of these. Regarding amplification time, the shortest amplification times were observed for guide probes (3'-OH)_1_OH and guide probe (3'-blocked) with amino-modified 3' ends. Figure 17 ).

[0440] Experimental Conclusions: 3' end modification of the guide probe has a certain impact on Taq DNA polymerase digestion activity. Guide probes with a 3' amino terminus (3'-blocked) exhibited similar amplification time and fluorescence signal to guide probes with a 3'-OH terminus, which may be a new option in guide probe design for Taq DNA polymerase. Overall, 3'-blocked guide probes could activate Taq DNA polymerase digestion activity to varying degrees. Due to the superior performance of amino modification, 3'-blocked guide probes were used in subsequent experiments.

[0441] Example 12. Effect of the 3' end base type of the guide probe (3'-block) on Taq DNA polymerase digestion activity

[0442] Objective: To investigate the effect of the 3' end base type of the guide probe (3'-block) on the digestion activity of Taq DNA polymerase.

[0443] Experimental Design: The experimental design is the same as in "Example 10". The guide probes (3'-closed) designed in this example are all guide probes (3'-closed) with 0 bases overlapping at the 3' end.

[0444] The reaction components and conditions were the same as in "Example 10". The oligonucleotides used are shown in Table 10. The 3' terminal bases of the guide probes (3'-blocking) used in the experiment were A, T, C, and G, with the 3' terminal base being T, which matched the template.

[0445] Table 10. Sequences Used

[0446]

[0447] Experimental results ( Figure 18The reaction fluorescence signal of the guide probe with a 3' end base complementary to the template (3'-block) was the highest, which was higher than that of all guide probes with a 3' end base not complementary to the template (3'-block).

[0448] Experimental conclusion: The type of the 3' end base of the guide probe (3'-block) affects the digestion activity of Taq DNA polymerase, and for the blocked guide probe (3'-block), the 3' end base is complementary to the template, which is the preferred design.

[0449] Example 13. Effect of the number of overlapping bases at the 3' end of the guide probe (3'-block) on Taq DNA polymerase digestion activity.

[0450] Objective: To investigate the effect of the number of overlapping bases at the 3' end of the guide probe (3'-block) on the digestion activity of Taq DNA polymerase.

[0451] Experimental design: The experimental design is the same as in "Example 9".

[0452] The reaction components and reaction conditions are the same as in "Example 9". The oligonucleotides used are shown in Table 11.

[0453] Table 11. Sequences Used

[0454]

[0455]

[0456] Experimental results: In the reaction with the addition of a guide probe (3'-blocking), the best results and the highest fluorescence signal were observed when the 3' terminal base overlapped with the downstream double strand by 0 bases. Figure 19 ).

[0457] Experimental conclusion: The number of overlapping bases at the 3' end of the guide probe (3'-block) affects the digestion activity of Taq DNA polymerase, and for the guide probe (3'-block), the preferred design is that the 3' end base overlaps with the downstream double strand by 0 bases (and this base is complementary to the template).

[0458] Example 14. Application of the guide probe (3'-closed) – Introduction of a guide probe (3'-closed) into the LAMP-Taq real-time detection system

[0459] Experimental objective: To investigate the effect of the guide probe (3'-closed) on the LAMP-Taq real-time detection system.

[0460] Experimental Design: The experimental design is the same as in "Example 6". Based on the scheme described in Example 6, two sets of parallel experiments were conducted using guide probes (3'-blocking) and guide probes (3'-OH) respectively to compare the effects of guide probes (3'-blocking) and guide probes (3'-OH) on the LAMP-Taq real-time detection system.

[0461] The guide probe (3'-blocked) used in this experiment was designed with zero base overlap at the 3' end and complementary to the template. The remaining reaction components and reaction conditions were the same as in "Example 6". The oligonucleotides used are shown in Table 12.

[0462] Table 12: Sequences Used

[0463]

[0464]

[0465] Experimental results ( Figure 20 Regarding amplification time, in the LAMP-Taq real-time detection system, the reaction with the guide probe (3'-block) reaches the detection threshold in a shorter time than the reaction with the guide probe (3'-OH).

[0466] Experimental conclusion: Introducing a guide probe with zero base overlap at the 3' end (3'-blocked) into the LAMP-Taq real-time detection system is effective and superior to a guide probe with one base overlap at the 3' end (3'-OH).

[0467] Example 15. Application of the guide probe (3'-closed) – Identification of SNPs in a LAMP-Taq real-time detection system

[0468] Experimental objective: The experimental principle is as follows. Figure 21 The study investigated the performance of the guide probe (3'-closed) in distinguishing mutations in the LAMP-Taq real-time detection system.

[0469] Experimental Design: The experimental design is the same as in "Example 6". Based on the scheme described in Example 6, two sets of parallel experiments were conducted using guide probe (3'-blocking) and guide probe (3'-OH) respectively to compare the ability of guide probe (3'-blocking) and guide probe (3'-OH) to distinguish mutations in the LAMP-Taq real-time detection system.

[0470] The guide probe (3'-blocked) used in this experiment was designed with the 3' terminal base overlapping the downstream double strand by 0 bases. The remaining reaction conditions and components were the same as in "Example 6". The oligonucleotides used are shown in Table 13.

[0471] Table 13: Sequences Used

[0472]

[0473]

[0474] Experimental results: Only the reaction with wild-type template produced a fluorescent signal; the reactions with the three mutant templates did not produce a fluorescent signal. Figure 22 ).

[0475] Experimental conclusion: Adding a guide probe (3'-blocking) to the LAMP-Taq real-time detection system can achieve complete mutation differentiation, but when using a guide probe (3'-OH), the LAMP-Taq real-time detection system cannot completely differentiate mutations. Figure 23 This indicates that, for Taq DNA polymerase, the guide probe (3'-block) is superior to the guide probe (3'-OH) in terms of enzyme digestion specificity.

[0476] Example 16. Mass spectrometry verification of the restriction enzyme specificity of Taq DNA polymerase

[0477] Experimental objective: To investigate the digestion characteristics of Taq DNA polymerase under the action of guide probe (3'-OH) / guide probe (3'-block) using MALDI-TOF mass spectrometry.

[0478] Experimental Design: The experimental design is the same as in "Example 3". Based on the guide probe (3'-blocking)_2, guide probe (3'-OH)_2, and first oligonucleotide probe_1, four target sequences were designed and synthesized: target sequence_WT, target sequence_MUT_1, target sequence_MUT_2, and target sequence_MUT_3. The 5' nucleotide (C) of the second target-specific sequence of the first oligonucleotide probe_1 is complementary to the nucleotide (G) of the target sequence_WT, but not complementary to target sequences_MUT_1, target sequence_MUT_2, and target sequence_MUT_3.

[0479] The reaction components are as follows: 0.2 μM target sequence _WT / MUT_1 / 2 / 3, 0.5 μM guide probe (3'-blocking)_2 / guide probe (3'-OH)_2, 0.5 μM first oligonucleotide probe_1, 1×thermol buffer (20 mM Tris-HCl, 10 mM (NH4)2SO4, 150 mM KCl, 2 mM MgSO4, 0.1%) 20 (pH 8.8 at 25℃), 6.0 mM MgCl2 and 1.0 UTaq01 enzyme, and sterile water were added to a final volume of 10 μL. The reaction was performed on a SLAN-96S fluorescent PCR instrument (Shanghai Hongshi Medical Technology Co., Ltd., China) at 65℃ for 50 minutes. Mass spectrometry of the products was performed on a CP-Light 1000 microbial mass spectrometry detection system (Xiamen Jinnuohua Scientific Instruments Co., Ltd., China). The oligonucleotides in the reaction were synthesized by Shanghai Bioengineering Co., Ltd. (Table 14):

[0480] Table 14: Sequences Used

[0481]

[0482] Experimental results:

[0483] (1) Under the action of the guide probe (3'-closed), a signal peak corresponding to the mediator subsequence was observed only in the response of the target sequence _WT (confirming successful WT cleavage).

[0484] No signal peaks corresponding to the mediator subsequence were observed in the reactions of _MUT1 / 2 / 3. Figure 24 );

[0485] (2) Under the action of the guide probe (3'-block), no complete signal peak corresponding to Flap_Probe_1 was observed only in the reaction of the target sequence _WT, while complete signal peaks corresponding to Flap_Probe_1 were observed in the reactions of the target sequences _MUT1 / 2 / 3 (confirming that MUT1 / 2 / 3 were not cleaved). Figure 24 ).

[0486] (3) Under the action of the guide probe (3'-OH), signal peaks corresponding to the mediator subsequences could be observed in the reactions of both the target sequence _WT and the target sequence _MUT1 / 2 / 3. Figure 25 );

[0487] (4) Under the action of the guide probe (3'-OH), no complete signal peak corresponding to Flap_Probe_1 was observed in the reactions of target sequence _WT and target sequence _MUT1 / 2 / 3. Figure 25 ).

[0488] Experimental conclusions: Mass spectrometry results demonstrate that, with the aid of the upstream primer, the guide probe (3'-blocked), and the first oligonucleotide probe_1 (whose 5' end nucleotide of the second target-specific sequence is complementary to the target nucleic acid), Taq DNA polymerase exhibits higher enzymatic specificity and can specifically cleave oligonucleotides. However, with the aid of the guide probe (3'-OH), Taq DNA polymerase cannot specifically recognize the site and is prone to non-specific cleavage.

[0489] Example 17. Application of the guide probe (3'-closed) – Introduction of a guide probe (3'-closed) into the CPA-fluorescent probe real-time detection system

[0490] Experimental objective: To investigate the effect of introducing a guide probe (3'-blocking) into the CPA-fluorescent probe real-time detection system.

[0491] Experimental Design: The CPA reaction system used in the experiment was the same as in "Example 4". Based on the established CPA system, fluorescent probe 1 and the corresponding guide probe (3'-blocking) 3 were designed.

[0492] The experiment consisted of two reactions. Reaction 1 was composed of the following components: 0.2 μM fluorescent probe 1, 0.2 μM guide probe (3'-blocking) 3, 0.8 μM CPA_N_1s, 0.4 μM CPA_N_2a / 3a, 0.5 μM CPA_N_4s, 0.2 μM CPA_N_5a, 1×thermol buffer (20 mM Tris-HCl, 10 mM (NH4)2SO4, 150 mM KCl, 2 mM MgSO4, 0.1%). 20 (pH 8.8 at 25℃), 6.0 mM MgCl2, 8.0 U BST Polymerase, 1.0 U Taq Polymerase, and 5 μL template DNA were added, and sterile water was added to a final volume of 25 μL. Reaction 2 had the same components as reaction 1 but without the guide probe (3'-blocking)_3. The reaction was performed on a SLAN-96S fluorescent PCR instrument (Shanghai Hongshi Medical Technology Co., Ltd., China), set at 65℃ for 50 minutes, with fluorescence detected simultaneously. The oligonucleotides in the reaction were synthesized by Shanghai Bioengineering Co., Ltd. (Table 15).

[0493] Table 15: Sequences Used

[0494]

[0495] Experimental results: Compared with reaction 2 without the guide probe (3'-blocking) as a control, reaction 1 with the guide probe (3'-blocking) showed a higher fluorescence signal and a shorter amplification time to reach the detection threshold. Figure 26 ).

[0496] Experimental conclusion: Adding a guide probe (3'-blocking) to the CPA-fluorescent probe system can further improve the signal of the fluorescent probe detection system and shorten the reaction time. This may indicate the emergence of a brand-new method to improve the signal of the fluorescent probe detection system, which can be used in different fluorescent probe detection systems for signal enhancement.

[0497] Example 18. Application of the guide probe (3'-closed) – Introduction of a guide probe (3'-closed) into the LAMP-fluorescent probe real-time detection system

[0498] Experimental objective: To investigate the effect of introducing a guide probe (3'-blocking) into a LAMP-fluorescent probe real-time detection system.

[0499] Experimental Design: The LAMP reaction system used in this experiment is the same as in "Experiment 10". Based on the established LAMP system, fluorescent probe 2 and the corresponding guide probe (3'-blocking) 4 were designed.

[0500] The experiment consisted of two reactions. Reaction 1 consisted of the following components: 0.2 μM fluorescent probe 2, 0.2 μM guide probe (3'-blocking) 4, 0.2 μM LAMP_N_F3 / B3, 2 μM LAMP_N_FIP / BIP, 0.4 μM LAMP_N_LF / BF, 0.5 μM guide probe (3'-OH) 2, 1×thermol buffer (20 mM Tris-HCl, 10 mM (NH4)2SO4, 150 mM KCl, 2 mM MgSO4, 0.1%). 20 (pH 8.8 at 25℃), 2mM MgCl2, 1mM dNTPs, 8.0U BST Polymerase, 1.0U Taq Polymerase, and 5μL template DNA were added, and sterile water was added to a final volume of 25μL. Reaction 2 had the same components as reaction 1 but without the guide probe (3'-blocking)_4. The reactions were performed on a SLAN-96S fluorescent PCR instrument (Shanghai Hongshi Medical Technology Co., Ltd., China), at a temperature of 65℃ for 50 minutes, with fluorescence detected simultaneously. The oligonucleotides used in the reactions were synthesized by Shanghai Bioengineering Co., Ltd. (Table 16).

[0501] Table 16: Sequences Used

[0502]

[0503] Experimental results: Compared with reaction 2 without the guide probe (3'-blocking), reaction 1 with the guide probe (3'-blocking) showed a significantly higher fluorescence signal, a shorter amplification time to reach the detection threshold, and a tenfold increase in sensitivity. Figure 27 ).

[0504] Experimental conclusion: Adding a guide probe (3'-blocking) to the LAMP-fluorescent probe detection system can further enhance the signal and shorten the reaction time. This demonstrates that this method can be used for signal enhancement in different fluorescent probe detection systems, and it has been validated in CPA, LAMP, and even PCR.

[0505] Example 19. Effect of the guide probe (3'-OH) on Mediator Probe PCR signal

[0506] Experimental objective: After the probe in Mediator Probe PCR binds to the template, the mediator sequence can be cleaved. Therefore, we further investigated the effect of introducing a guide probe (3'-OH) into Mediator Probe PCR.

[0507] Experimental Design: Taking the K12 antigen gene in *E. coli* as an example, the following primers were designed: upstream primer (K12_F), downstream primer (K12_R), first oligonucleotide probe (K12_MP_1), second oligonucleotide probe (Mediator UR_1), and guide probe (3'-OH) (K12_guide probe (3'-OH)). The experimental reaction system was as follows: 1×PCR buffer (7mM Tris-HCl, 16.6mM (NH4)2SO4, 6.7μM EDTA and 0.085mg / mL BSA), 7.0mM MgCl2, 0.2mM dNTPs, 3.0U of polymerase Taq, 40nM upstream primer, 40nM downstream primer, 20nM first oligonucleotide probe, 40nM second oligonucleotide probe, and 20nM guide probe (3'-OH) or deionized water. The reaction conditions were: 95℃ for 5 min; followed by 45 cycles (95℃ for 15 s and 60℃ for 45 s); fluorescence was collected at 60℃. The experimental instrument used was the SLAN-96S fluorescence quantitative PCR instrument from Shanghai Hongshi Medical Technology Co., Ltd., China. The oligonucleotides used were synthesized by Shanghai Bioengineering Co., Ltd. (Table 17).

[0508] Table 17: Oligonucleotides used

[0509]

[0510]

[0511] Experimental results: The detection signal intensity was improved after adding the guide probe (3'-OH). Figure 28 ).

[0512] Experimental conclusion: Adding a guide probe (3'-OH) to Mediator Probe PCR can significantly enhance signal intensity.

[0513] Example 20. Effect of guide probe (3'-blocked) on Mediator Probe PCR signal

[0514] Objective: To investigate the effect of guide probes (3'-blocked) on detection signals in Mediator Probe PCR.

[0515] Experimental Design: Taking the K12 antigen gene from *E. coli* (GenBank ID: CP117020.1) as an example, an upstream primer (K12_F), a downstream primer (K12_R), a first oligonucleotide probe (K12_MP_1), a second oligonucleotide probe (Mediator UR_1), and a guide probe (3'-blocking) (K12_Guide) were designed. The reaction components, reaction conditions, and instruments were the same as in "Example 20". The oligonucleotides used were synthesized by Shanghai Bioengineering Co., Ltd. (Table 18).

[0516] Table 18: Practical Oligonucleotides

[0517]

[0518] Experimental results: The detection signal intensity of Mediator Probe PCR was improved after the addition of the guide probe (3'-blocking). Figure 29 ).

[0519] Experimental conclusion: Adding a guide probe (3'-block) to Mediator Probe PCR can also significantly increase signal intensity.

[0520] Example 21. Effect of the guide probe (3'-OH) on the signal of the MeltArray single detection system

[0521] Experimental objective: After the first oligonucleotide probe in MeltArray binds to the template, the mediator sequence can be cleaved. Therefore, we further investigated the effect of introducing a guide probe (3'-OH) into the MeltArray single detection system.

[0522] Experimental design: Taking the H6 and Yamagata genes of influenza virus as examples, upstream primers (H6_F, Yamagata_F), downstream primers (H6_R, Yamagata_R), first oligonucleotide probes (H6_MP-1, Yamagata_MP_1), second oligonucleotide probes (FAM_UR_L, FAM_UR_H, Cy5_UR_L and Cy5_UR_H) were designed, and a guide probe (3'-OH) was designed for investigation. The experimental reaction system was as follows: 1×PCR buffer (7mM Tris-HCl, 16.6mM (NH4)2SO4, 6.7μM EDTA and 0.085mg / mL BSA), 7.0mM MgCl2, 0.2mM dNTPs, 3.0U Taq polymerase, 40nM second oligonucleotide probes FAM_UR_L, FAM_UR_H, Cy5_UR_L and Cy5_UR_H, 40nM upstream primer, 40nM downstream primer, 40nM first oligonucleotide probe, and 40nM guide probe (3'-OH) or deionized water. The reaction conditions were: 95℃ for 5 min; then 50 cycles (95℃, 20s and 60℃, 1 min); 35℃ for 40 min; followed by fluorescence collection during the 45℃-95℃ melting process. The experimental instruments used were the same as in "Example 19". The oligonucleotides used were synthesized by Shanghai Bioengineering Co., Ltd. (Table 19):

[0523] Table 19: Oligonucleotides used

[0524]

[0525] Experimental results: Introducing a guide probe (3'-OH) into the MeltArray single detection system resulted in signal enhancement across different targets. Figure 30 ).

[0526] Experimental conclusion: Introducing a guide probe (3'-OH) into the MeltArray single detection system can enhance the melting peak signal.

[0527] Example 22. Effect of Guide Probes (3'-OH) on the Signal of MeltArray Multiplex Detection System. Experimental objective: To investigate the effect of introducing multiple object guide probes (3'-OH) into the MeltArray multiplex detection system.

[0528] Experimental Design: The following genes from influenza virus were used: H1 (GenBank: MN594886.1), H5 (GenBank: CY040899.1), H10 (GenBank: JX500443), H12 (GenBank: MH134690), H18 (GenBank: CY125945), Yamagata (GenBank: OQ034432), N2 (GenBank: CY005306.1), and N11 (GenBank: MH682). Using the following genes as examples (205), we designed upstream primers, downstream primers, a first oligonucleotide probe, a second oligonucleotide probe, and guide probes (3'-OH) for each target for investigation: H3 gene (GenBank: KJ577143.1), H4 gene (GenBank: MN911292.1), H6 gene (GenBank: OQ054018.1), H7 gene (GenBank: MH209475.1), H11 gene (GenBank: MF146469), and N4 gene (GenBank: CY003986.1). The experimental reaction components, conditions, and instruments were the same as in "Example 20". The oligonucleotides used were synthesized by Shanghai Bioengineering Co., Ltd. (Table 20).

[0529] Table 20: Oligonucleotides used

[0530]

[0531]

[0532]

[0533] Experimental results: In the MeltArray multiplex detection system, without the introduction of the guide probe (3'-OH), all 26 target melting peaks rose normally; after the introduction of the guide probe (3'-OH), one melting peak signal was significantly enhanced, two target melting peaks showed no significant change, 11 melting peak signals were significantly reduced, and 12 melting peak signals disappeared. Figure 31 ).

[0534] Experimental conclusion: The guide probe (3'-OH) produces severe nonspecificity in the MeltArray multiplex detection system, resulting in signal reduction or disappearance of most melting peaks.

[0535] Example 23. Upper limit of the number of targets detected by the MeltArray multiplex detection system under the action of the guide probe (3'-OH)

[0536] Experimental objective: To investigate the effect of gradually increasing the number of targets on the detection signal after introducing a guide probe (3'-OH) into the MeltArray multiplex detection system.

[0537] Experimental Design: The following genes from influenza virus were used: H1 (GenBank: MN594886.1), H5 (GenBank: CY040899.1), H10 (GenBank: JX500443), H12 (GenBank: MH134690), H18 (GenBank: CY125945), Yamagata (GenBank: OQ034432), N2 (GenBank: CY005306.1), and N11 (GenBank: MH68). Using the following genes as examples (2205), H3 (GenBank: KJ577143.1), H4 (GenBank: MN911292.1), H6 (GenBank: OQ054018.1), H7 (GenBank: MH209475.1), H11 (GenBank: MF146469), and N4 (GenBank: CY003986.1), upstream primers, downstream primers, a first oligonucleotide probe, a second oligonucleotide probe, and a guide probe (3'-OH) were designed for investigation. The experimental reaction components, conditions, and instruments were the same as in "Example 20". The oligonucleotides used were synthesized by Shanghai Bioengineering Co., Ltd. (Table 21).

[0538] Table 21: Oligonucleotides used

[0539]

[0540]

[0541]

[0542] Experimental results: Introducing a guide probe (3'-OH) into a detection system with fewer targets (target number < 10) also enhances the detection signal of the target. However, introducing more guide probes (3'-OH) into a detection system with more targets (target number > 10) actually reduces the detection signal of the target. Figure 32 ).

[0543] Experimental conclusion: In multiplex detection systems, the use of a guide probe (3'-OH) may result in non-specific hybridization with oligonucleotides in the system and the formation of dimers, which may affect the detection of specific melting peaks.

[0544] Example 24. Effects of different base overlap numbers and 3'-terminal modified guide probes (3'-blocked) on the signal of the MeltArray single detection system.

[0545] Experimental objective: To investigate the number of base overlaps and 3' end modifications of guide probes (3'-blocking) suitable for MeltArray.

[0546] Experimental Design: Taking the O4 antigen gene (GenBank: CP051113.1) from Vibrio parahaemolyticus as an example, upstream primer (O4_F), downstream primer (O4_R), first oligonucleotide probe (O4_MP_1), second oligonucleotide probe (ROX_UR_L and ROX_UR_H) were designed. The guide probe (3'-blocking) was designed with 3' end base matching and 0 or 1 base overlap. 3' end modifications were selected from 3'-P, 3'-C3 Spacer, and 3'-NH2 C7. The combination of base overlap and 3' end modification was investigated. The experimental reaction components, conditions, and instruments were the same as in "Example 20". The oligonucleotides used were synthesized by Shanghai Bioengineering Co., Ltd. (Table 22).

[0547] Table 22: Oligonucleotides used

[0548]

[0549]

[0550] Experimental results: Guide probes with zero overlapping bases (3'-blocked) all enhanced the melting peak, with the guide probe with zero overlapping bases and 3'-NH2 C7 modified at the 3' end (3'-blocked) showing the most significant signal enhancement. Figure 33 ).

[0551] Experimental conclusion: The guide probe (3'-blocked) with zero overlapping bases and 3' end modified with 3'-NH2 C7 has a better ability to enhance the melting peak signal in MeltArray. Therefore, the design of related guide probes (3'-blocked) in MeltArray in the future should be zero overlapping bases and 3' end modified with 3'-NH2 C7.

[0552] Example 25. Effect of guide probe (3'-blocking) concentration on the signal of the MeltArray single detection system

[0553] Experimental objective: To investigate the effect of the concentration of the guide probe (3'-blocking) on ​​the melting peak height in this system.

[0554] Experimental Design: Taking the O4 antigen gene (GenBank: CP051113.1) from Vibrio parahaemolyticus as an example, upstream primers, downstream primers, a first oligonucleotide probe, a second oligonucleotide probe, and a guide probe (3'-blocking) were designed for investigation. The final concentrations of the guide probe (3'-blocking) were: 0 nM (control without guide probe (3'-blocking)), 10 nM, 20 nM, 40 nM, and 80 nM. The experimental reaction components, conditions, and procedures were the same as in "Example 20". The oligonucleotides used were synthesized by Shanghai Bioengineering Co., Ltd. (Table 23).

[0555] Table 23: Oligonucleotides used

[0556]

[0557]

[0558] Experimental results: With increasing concentration of the guide probe (3'-blocking), no nonspecific [effects] were observed in the system, and higher concentrations of template in the wells (10) did not result in [a specific effect]. 3 -10 2 The melting peak of the template pores (copies / μL) showed a gradually increasing trend, while the melting peak of the template pores at lower concentrations (10 copies / μL) showed a gradually increasing trend. 1 -10 0 The enhancement effect on the melting peak in copies / μL was relatively weak. Figure 34 ).

[0559] Experimental conclusions: In principle, increasing the concentration of the guide probe (3'-blocking) will not cause non-specific signals to the system, and the enhancement effect on the melting peak also increases with the increase of the guide probe (3'-blocking) concentration; the enhancement effect is more obvious for higher concentration templates.

[0560] Example 26. Upper limit of the number of targets detected by the MeltArray multiplex detection system under the action of the guide probe (3'-closed)

[0561] Experimental objective: To investigate the effect of gradually increasing the number of targets on the signal when a guide probe (3'-closed) is added to the MeltArray multiplex detection system.

[0562] Experimental Design: The following genes from influenza virus were used: H1 (GenBank: MN594886.1), H5 (GenBank: CY040899.1), H10 (GenBank: JX500443), H12 (GenBank: MH134690), H18 (GenBank: CY125945), Yamagata (GenBank: OQ034432), N2 (GenBank: CY005306.1), and N11 (GenBank: M... Using the following genes as examples: H682205, H3 (GenBank: KJ577143.1), H4 (GenBank: MN911292.1), H6 (GenBank: OQ054018.1), H7 (GenBank: MH209475.1), H11 (GenBank: MF146469), and N4 (GenBank: CY003986.1), upstream primers, downstream primers, a first oligonucleotide probe, a second oligonucleotide probe, and a guide probe (3'-blocking) were designed. The experimental reaction components, conditions, and instruments were the same as in "Example 20". The oligonucleotides used were synthesized by Shanghai Bioengineering Co., Ltd. (Table 24).

[0563] Table 24: Oligonucleotides used

[0564]

[0565]

[0566] Experimental results: Adding a guide probe (3'-closed) to a detection system with fewer targets (target number < 10) enhances the signal of the target; adding a guide probe (3'-closed) to a detection system with more targets (target number > 10) still enhances the signal. Figure 35 ).

[0567] Experimental conclusion: The application of the guide probe (3'-closed) in the MeltArray multiplex detection system is not limited by the number of targets to be detected, and it can still significantly improve the signal in multiplex detection systems.

[0568] Example 27. Effect of the guide probe (3'-closed) on the signal of the MeltArray multiplex detection system

[0569] Experimental objective: To investigate the impact of introducing multiple object guide probes (3'-closed) into the MeltArray multi-detection system.

[0570] Experimental Design: The following genes from influenza virus were used: H1 (GenBank: MN594886.1), H5 (GenBank: CY040899.1), H10 (GenBank: JX500443), H12 (GenBank: MH134690), H18 (GenBank: CY125945), Yamagata (GenBank: OQ034432), N2 (GenBank: CY005306.1), and N11 (GenBank: M... Using the following genes as examples: H682205, H3 (GenBank: KJ577143.1), H4 (GenBank: MN911292.1), H6 (GenBank: OQ054018.1), H7 (GenBank: MH209475.1), H11 (GenBank: MF146469), and N4 (GenBank: CY003986.1), upstream primers, downstream primers, a first oligonucleotide probe, a second oligonucleotide probe, and a guide probe (3'-blocking) were designed. The experimental reaction components, conditions, and instruments were the same as in "Example 20". The oligonucleotides used were synthesized by Shanghai Bioengineering Co., Ltd. (Table 25).

[0571] Table 25: Oligonucleotides used

[0572]

[0573] Experimental results:

[0574] 1. In the MeltArray multiplex detection system, without the introduction of a guide probe (3'-blocking), all 26 target melting peaks rose normally. Figure 36 ).

[0575] 2. When a guide probe (3'-block) is introduced into the MeltArray multiplex detection system, all 26 melting peaks not only rise normally, but the signals are also enhanced. Figure 36 ).

[0576] 3. When a guide probe (3'-OH) was introduced into the MeltArray multiplex detection system, one melting peak signal was significantly enhanced, two target melting peaks showed no significant change, eleven melting peak signals were significantly reduced, and twelve melting peak signals disappeared. Figure 36 ).

[0577] Experimental conclusion: The guide probe (3'-block) is more suitable for multiple detection systems than the guide probe (3'-OH). It not only has the ability to stably enhance the signal, but also does not generate non-specific signals in complex systems with multiple targets.

[0578] Example 28. Effect of the guide probe (3'-closed) on the sensitivity of the MeltArray system

[0579] Experimental objective: To investigate the effects of introducing a guide probe (3'-closed) into the MeltArray on other aspects of the system, starting with the effect on the system's sensitivity.

[0580] Experimental Design: Taking the O5 antigen gene of Vibrio parahaemolyticus (GenBank: JQ863078.1) as an example, upstream primers, downstream primers, a first oligonucleotide probe, a second oligonucleotide probe, and a guide probe (3'-blocked) were designed. First, the guide probe (3'-blocked) of the O5 antigen gene was added to the singlet to investigate singlet sensitivity. Then, the O5 antigen gene (GenBank: JQ863078.1), O8 antigen gene (GenBank: CP046761.1), O1 antigen gene (GenBank: CP034305.1), O7 antigen gene (GenBank: CP046806.1), O11 antigen gene (GenBank: CP085857.1), O2 antigen gene (GenBank: MK503852.1), and O3 / Multiplex sensitivity studies were conducted using a guide probe (3'-blocked) of the O5 antigen gene in multiplexed samples of the O13 antigen gene (GenBank: CP046763.1), O4 antigen gene (GenBank: CP051113.1), O10-1 antigen gene (GenBank: JQ863076.1), O10-2 antigen gene (GenBank: KT459785.1), and O9 antigen gene (GenBank: CP110670.1). The experimental reaction components, conditions, and instruments were the same as in "Example 20". The oligonucleotides used were synthesized by Shanghai Bioengineering Co., Ltd. (Table 26).

[0581] Table 26: Oligonucleotides used

[0582]

[0583]

[0584]

[0585] Experimental results:

[0586] 1. In a single weight, the O5 gene has 10 [units / items]. 1 -10 0 The melting peaks of copies / μL template wells all rose, and the signal was enhanced after the introduction of the guide probe (3'-closed). Figure 37 ).

[0587] 2. In multiples, the O5 gene is 10. 1 The melting peak rises at copies / μL template wells, and the signal is enhanced after the introduction of the guide probe (3'-closed), 10 0 No signal was observed at copies / μL, consistent with the control group. Figure 38 ).

[0588] Experimental conclusion: Introducing a guide probe (3'-blocking) into the MeltArray can improve the melting peak of low-concentration templates.

[0589] Example 29. Effect of the guide probe (3'-closed) on the detection time of the MeltArray system

[0590] Experimental objective: To investigate the effects of introducing a guide probe (3'-closed) into the MeltArray on other aspects of the system, and to determine the effect of introducing a guide probe (3'-closed) into the MeltArray on the detection time of the system.

[0591] Experimental design: Vibrio parahaemolyticus O5 antigen gene (GenBank: JQ863078.1), O8 antigen gene (GenBank: CP046761.1), O1 antigen gene (GenBank: CP034305.1), O7 antigen gene (GenBank: CP046806.1), O11 antigen gene (GenBank: CP085857.1), O2 antigen gene (GenBank: MK503852.1), and O3 / O13 antigen gene (GenBank: CP046763) were used respectively. 1) Taking the O4 antigen gene (GenBank: CP051113.1), O10-1 antigen gene (GenBank: JQ863076.1), O10-2 antigen gene (GenBank: KT459785.1), and O9 antigen gene (GenBank: CP110670.1) as examples, upstream primers, downstream primers, a first oligonucleotide probe, a second oligonucleotide probe, and a guide probe (3'-block) were designed to investigate the duration of the mediator sequence extension program segment (35℃). The experimental reaction components and instruments were the same as in "Example 20". The reaction conditions were: 95℃ for 5 min; then 50 cycles (95℃, 20 s and 60℃, 1 min); 35℃ for 40 min, 20 min, or 10 min were set; fluorescence was collected simultaneously during the melting process from 45℃ to 95℃. The oligonucleotides used were synthesized by Shanghai Bioengineering Co., Ltd. (Table 27).

[0592] Table 27: Oligonucleotides used

[0593]

[0594]

[0595] Experimental results:

[0596] 1. Introducing a guide probe (3'-blocking) into the entire system increases the melting peaks of all components, while shortening the duration of the mediator subsequence extension segment (35℃) decreases the melting peaks. Figure 39 ).

[0597] 2. Among them, the O8 antigen is significantly affected by the shortened detection time, with a marked decrease in the melting peak. Therefore, taking the O8 antigen as an example for analysis: the melting peak height at the original time (40 min) without the introduction of the guide probe (3'-blocking) is similar to the melting peak height at the shortened time (10 min) with the introduction of the guide probe (3'-blocking). Therefore, the introduction of the guide probe (3'-blocking) in this system can shorten the detection time by 30 min. Figure 40 ).

[0598] Experimental conclusion: Introducing a guide probe (3'-closed) into a multi-system can theoretically reduce the detection time of the system. In this system, it can reduce the time by about 30 minutes. However, when the target and dosage are different in different systems, the upper and lower limits of the time reduction need to be investigated separately. The results of this experiment are for reference only.

[0599] Example 30. Effect of guide probe (3'-closed) on the detection length of MeltArray amplicon.

[0600] Experimental objective: To investigate the effects of introducing a guide probe (3'-closed) into the MeltArray on other aspects of the system and to determine its impact on the detection length of amplicon.

[0601] Experimental Design: Taking the RPP30 gene (GenBank: NC_000010.11) in the human genome as an example, the binding regions of the downstream primer and the first oligonucleotide probe were fixed. Amplicons of different lengths were investigated by designing upstream primers at different positions. Amplicons of lengths of 397bp, 979bp, 1500bp, 2019bp, 2266bp, 2468bp, and 2674bp were examined. Different upstream primers, downstream primers (RPP30_R), first oligonucleotide probes (RPP30_FAM_MP), second oligonucleotide probes (FAM_UR_L, FAM_UR_H), and their corresponding guide probes (3'-blocking) (RPP30_MP_Guide) were designed. The experimental reaction components, conditions, and instruments were the same as in "Example 20". The oligonucleotides used were synthesized by Shanghai Bioengineering Co., Ltd. (Table 28).

[0602] Table 28: Oligonucleotides used

[0603]

[0604] Experimental results: The probe showed a decreasing trend in melting peak when the amplicon length increased to 979 bp, while the actual amplicon length of 2266 bp basically had no melting peak signal; however, after adding the guide probe (3'-blocking), the melting peak only decreased significantly when the amplicon length increased to 2266 bp, and there was still a relatively obvious fluorescence signal when the amplicon length increased to about 2468 bp.

[0605] Experimental conclusion: Introducing a guide probe (3'-blocking) into MeltArray can improve the upper limit of amplicon detection from about 1000bp to about 2500bp, thus broadening the detection range of MeltArray. Figure 41 Furthermore, the guide probe (3'-block) can improve the amplification efficiency of the upstream primer.

[0606] Example 31. Effect of upstream primer and guide probe (3'-closed) partial overlap on the signal of the MeltArray single detection system

[0607] Experimental objective: To investigate the effect of partial overlap between the upstream primer and the guide probe (3'-closed) on the signal of the MeltArray single detection system.

[0608] Experimental design: Taking the V422del mutation site of the ESR1 gene in breast cancer (GenBank: NG_008493) as an example, a guide probe (3'-blocked) with 0 base overlap at the 3' end was designed, an upstream primer (V422del_F) partially overlapped with the guide probe (3'-blocked) sequence (15 nucleotide overlap), a downstream primer (V422del_R), a first oligonucleotide probe (V422del_MP), and a second oligonucleotide probe (HEX_UR) were designed. The experimental reaction system was as follows: 1×PCR buffer (7mM Tris-HCl, 16.6mM (NH4)2SO4, 6.7μM EDTA and 0.085mg / mL BSA), 6.0mM MgCl2, 0.2mM dNTPs, 2.0U of polymerase Taq, 40nM upstream primer, 40nM downstream primer, 40nM first oligonucleotide probe, 20nM second oligonucleotide probe, 40nM guide probe (3'-blocking) or deionized water. The reaction conditions were: 95℃, 2min; then 50 cycles (95℃, 15s and 60℃, 35s); 35℃, 30min; 95℃, 2min; 45℃, 2min; followed by fluorescence collection during the 45℃-95℃ melting process. The oligonucleotides used were synthesized by Shanghai Bioengineering Co., Ltd. (Table 29).

[0609] Table 29: Sequences Used

[0610]

[0611] Experimental results: In the MeltArray single detection system without the guide probe (3'-block), a melting peak signal with a certain height can be detected; after adding the guide probe (3'-block), the melting peak signal is significantly improved. Figure 42 ).

[0612] Experimental conclusion: Adding a guide probe (3'-closed) that partially overlaps with the upstream primer has a good effect on enhancing the melting peak signal of the MeltArray single detection system.

[0613] Example 32. Effect of complete overlap between upstream primer and guide probe (3'-closed) on the signal of the MeltArray single detection system

[0614] Experimental objective: To investigate the effect of complete overlap between the upstream primer and the guide probe (3'-closed) on the signal of the MeltArray single detection system.

[0615] Experimental Design: Taking the E380Q mutation site of the ESR1 gene (GenBank: NG_008493) in breast cancer as an example, a guide probe (3'-blocked) with 0 base overlap at the 3' end (E380Q_Guide), an upstream primer (E380Q_F) completely overlapping the sequence of the guide probe (3'-blocked), and a downstream primer (E380Q_R), a first oligonucleotide probe (E380Q_MP), and a second oligonucleotide probe (ROX_UR) were designed. The experimental instrument used was a SLAN-96S real-time PCR instrument from Shanghai Hongshi Medical Technology Co., Ltd. (China). The experimental reaction components, conditions, and instruments were the same as in "Example 31". The oligonucleotides used were synthesized by Shanghai Bioengineering Co., Ltd. (Table 30):

[0616] Table 30: Sequences Used

[0617]

[0618] Experimental results: In the MeltArray single detection system without the guide probe (3'-block), a melting peak signal with a certain height can be detected; after adding the guide probe (3'-block), the melting peak signal is significantly improved. Figure 43 ).

[0619] Experimental conclusion: Adding a guide probe (3'-blocked) that completely overlaps with the upstream primer has a good effect on enhancing the melting peak signal of the MeltArray single detection system.

[0620] In summary, regardless of the position of the upstream primer and the guide probe (3'-closed) (i.e., whether they overlap), the guide probe (3'-closed) can enhance the melting peak signal of the detection system.

[0621] Example 33. Effect of guide probe (3'-blocking) on ​​FEN1 cleavage activity

[0622] Objective: To investigate the effect of the guide probe (3'-block) on the FEN1 enzyme cleavage activity.

[0623] Experimental design: The experimental design is the same as in "Example 9", except that the Taq DNA polymerase in the reaction is replaced with FEN1 enzyme.

[0624] The reaction components and reaction conditions are the same as in "Example 9". The oligonucleotides used are shown in Table 31.

[0625] Table 31: Sequences Used

[0626]

[0627] Experimental results: No signal was generated in the reaction with the guide probe (3'-blocking), while a signal was generated in the reaction with the guide probe (3'-OH). Figure 44 ).

[0628] Experimental conclusion: The guide probe (3'-block) has a significant effect on the FEN1 enzyme cleavage activity. The guide probe (3'-block) is not suitable for FEN1 enzyme.

Claims

1. A method for detecting the presence of one or more target nucleic acids in a sample, the method comprising: (1) Provide one or more target nucleic acids to be detected, the target nucleic acids comprising a first region and a second region, wherein the first region is located downstream of the second region; For each target nucleic acid to be detected, the following are provided: At least one guide probe includes a first target-specific sequence comprising a sequence at least partially complementary to a first region of a target nucleic acid; optionally, the guide probe further includes a second sequence downstream of the first target-specific sequence; and At least one first oligonucleotide probe includes a mediator sequence and a second target-specific sequence in the 5' to 3' direction, the mediator sequence including a sequence that is not complementary to the target nucleic acid; and the second target-specific sequence including a sequence that is at least partially complementary to a second region of the target nucleic acid; Furthermore, under conditions that allow nucleic acid hybridization, the target nucleic acid is contacted with a guide probe and a first oligonucleotide probe; Preferably, the nucleotide at the 5' end of the second target-specific sequence is complementary to the second region of the target nucleic acid; preferably, the regions where the guide probe and the first oligonucleotide probe hybridize with the target nucleic acid are close (e.g., adjacent, adjacent to, or partially overlapping each other); (2) Under conditions that allow polymerase to cleave the first oligonucleotide probe, the product of step (1) is contacted with a polymerase having 5' nuclease activity. (3) Detect the product of step (2) (e.g., detect whether cleavage occurred in step (2); for example, detect whether one or more cleavage products exist in step (2)) to determine whether one or more target nucleic acids are present in the sample.

2. The method of claim 1, wherein, The guide probe does not contain a second sequence; Preferably, the first and second regions of the target nucleic acid are adjacent to each other; Preferably, when hybridizing with the target nucleic acid, the first target-specific sequence hybridizes in region A of the target nucleic acid, and the second target-specific sequence hybridizes in region B of the target nucleic acid; and, in the target nucleic acid, region A is located downstream of region B and adjacent to region B; Preferably, the interval between region A and region B is 5, 4, 3, 2, 1, or 0 nucleotides; preferably, the interval between region A and region B is 2, 1, or 0 nucleotides. Preferably, when the guide probe does not contain a second sequence, the nucleotides of the 3' portion (e.g., the 3' end) of the first target-specific sequence may be complementary to or not complementary to the target nucleic acid; Preferably, when the guide probe does not contain a second sequence, the first target-specific sequence has a 3'-OH end, or its 3' end is closed.

3. The method of claim 2, wherein, The 3' end of the first target-specific sequence is closed; Preferably, the 3' end of the guide probe is blocked by modifying the 3'-OH group of the nucleotide at the 3' end of the first target-specific sequence (e.g., by modifying an amino group or a phosphate group); or, the 3' end of the guide probe is blocked by adding a chemical moiety (e.g., biotin, alkyl, or a quencher group) to the 3'-OH group of the nucleotide at the 3' end of the first target-specific sequence; or, the 3' end of the guide probe is blocked by removing the 3'-OH group of the nucleotide at the 3' end of the first target-specific sequence; or, the 3' end of the guide probe is blocked by replacing the nucleotide at the 3' end of the first target-specific sequence with a dideoxynucleotide. Preferably, when the guide probe does not contain a second sequence, the 3' end of the guide probe is blocked by modifying the 3'-OH of the nucleotide at the 3' end of the first target-specific sequence with an amino group; Preferably, when the guide probe does not contain a second sequence, the region A and the region B are separated by 0 nucleotides; Preferably, when the guide probe does not contain a second sequence, the nucleotide at the 3' end of the first target-specific sequence is complementary to the target nucleic acid; Preferably, when the guide probe does not contain a second sequence, the nucleotide at the 3' end of the first target-specific sequence is complementary to the target nucleic acid, and the 3' end is closed (e.g., the nucleotide at the 3' end has an amino group modification); and the region A is separated from the region B by 0 nucleotides.

4. The method of claim 2, wherein, The first target-specific sequence has a 3'-OH terminus; Preferably, when the guide probe does not contain a second sequence, the nucleotide at the 3' end of the first target-specific sequence is not complementary to the target nucleic acid; Preferably, when the guide probe does not contain a second sequence, one or more nucleotides in the 3' portion of the first target-specific sequence are not complementary to the target nucleic acid; preferably, when the guide probe does not contain a second sequence, multiple consecutive nucleotides at the 3' end of the first target-specific sequence are not complementary to the target nucleic acid.

5. The method of claim 1, wherein, The guide probe also contains a second sequence downstream of the first target-specific sequence; Preferably, the first and second regions of the target nucleic acid are adjacent to each other; Preferably, when hybridizing with the target nucleic acid, the first target-specific sequence hybridizes in region A of the target nucleic acid, and the second target-specific sequence hybridizes in region B of the target nucleic acid; and, in the target nucleic acid, region A and region B are adjacent (e.g., separated by 0 nucleotides); Preferably, when hybridizing with the target nucleic acid, the second sequence is adjacent to the first target-specific sequence, and the length of the second sequence is 5, 4, 3, 2, or 1 nucleotide; preferably, the length of the second sequence is 3, 2, or 1 nucleotide. Preferably, when the guide probe further includes a second sequence downstream of the first target-specific sequence, the nucleotides of the 3' portion (e.g., the 3' end) of the second sequence may be complementary to or not complementary to the target nucleic acid; Preferably, when the guide probe further includes a second sequence downstream of the first target-specific sequence, the second sequence has a 3'-OH end, or its 3' end is closed; Preferably, when hybridizing with the target nucleic acid, the guide probe (e.g., the second sequence of the guide probe), the first oligonucleotide probe (e.g., the second target-specific sequence of the first oligonucleotide probe), and the target nucleic acid have partially overlapping sequences.

6. The method of claim 5, wherein, The 3' end of the second sequence is closed; Preferably, the 3' end of the guide probe is blocked by modifying the 3'-OH of the nucleotide at the 3' end of the second sequence (e.g., by modifying an amino group or a phosphate group); or, the 3' end of the guide probe is blocked by adding a chemical moiety (e.g., biotin, alkyl, or a quencher group) to the 3'-OH of the nucleotide at the 3' end of the second sequence; or, the 3' end of the guide probe is blocked by removing the 3'-OH of the nucleotide at the 3' end of the second sequence; or, the 3' end of the guide probe is blocked by replacing the nucleotide at the 3' end of the second sequence with a dideoxynucleotide. Preferably, when the guide probe contains a second sequence, the 3' end of the guide probe is blocked by modifying the 3'-OH of the nucleotide at the 3' end of the second sequence with an amino group; Preferably, when the guide probe hybridizes with the target nucleic acid, the second sequence is adjacent to the first target-specific sequence, and the length of the second sequence is 3, 2, or 1 nucleotide; Preferably, when the guide probe contains a second sequence, the nucleotide at the 3' end of the second sequence is complementary to the target nucleic acid.

7. The method of claim 5, wherein, The second sequence has a 3'-OH terminus; Preferably, when the guide probe hybridizes with the target nucleic acid, the second sequence is adjacent to the first target-specific sequence, and the length of the second sequence is 3, 2, or 1 nucleotide; Preferably, when the guide probe contains a second sequence, the nucleotide at the 3' end of the second sequence is not complementary to the target nucleic acid; Preferably, one or more nucleotides in the 3' portion of the second sequence are not complementary to the target nucleic acid; preferably, multiple consecutive nucleotides at the 3' end of the second sequence are not complementary to the target nucleic acid. Preferably, when the guide probe contains a second sequence, the nucleotide at the 3' end of the second sequence is not complementary to the target nucleic acid, and the nucleotide has a 3'-OH end; and, the region A and the region B are separated by 0 nucleotides, and the length of the second sequence is 1 nucleotide; Preferably, for each target nucleic acid to be detected, at least one upstream primer is also provided; wherein the upstream primer contains a sequence complementary to the target nucleic acid; and, when hybridizing with the target nucleic acid, the upstream primer is located upstream of the first target-specific sequence or has a sequence that at least partially overlaps with the first target-specific sequence; Preferably, after hybridization with the target nucleic acid, the upstream primer is located at the distal upstream end of the first target-specific sequence, or located adjacent upstream of the first target-specific sequence, or has a partially overlapping sequence with the first target-specific sequence (e.g., the 3' portion of the upstream primer has a partially overlapping sequence with the 5' portion of the first target-specific sequence), or has a completely overlapping sequence with the first target-specific sequence, or completely contains the first target-specific sequence. Preferably, in step (1), under conditions that allow nucleic acid amplification, the target nucleic acid is incubated with the upstream primer and an enzyme (e.g., polymerase); then, under conditions that allow nucleic acid hybridization, the guide probe and the first oligonucleotide probe are incubated with the amplification product to obtain the product of step (1); or, in step (1), under conditions that allow nucleic acid amplification and hybridization, the target nucleic acid is incubated with the upstream primer, enzyme, guide probe and the first oligonucleotide probe to obtain the product of step (1).

8. The method according to any one of claims 1-7, wherein, The method has one or more features selected from the following: (1) The sample contains either DNA, RNA, or a mixture of nucleic acids; (2) The target nucleic acid is DNA or RNA; and / or, the target nucleic acid is single-stranded or double-stranded; (3) The sample or target nucleic acid sequence is obtained from sources selected from the following: prokaryotes, eukaryotes, viruses or viroids; (4) The sample is selected from blood, saliva, urine, feces, cerebrospinal fluid, pleural fluid, milk, lymph, sputum, semen, or any combination thereof; (5) The target nucleic acid contains one or more SNP sites; (6) The length of the first region of the target nucleic acid is 11-150nt, for example, 11-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt, 80-90nt, 90-100nt, 100-110nt, 110-120nt, 120-130nt, 130-140nt, 140-150nt; (7) In step (1), provide 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more target nucleic acids to be detected.

9. The method according to any one of claims 1-8, wherein, The polymerase having 5' nuclease activity is a nucleic acid polymerase (e.g., DNA polymerase, especially a thermostable DNA polymerase) having 5' nuclease activity (e.g., 5' exonuclease activity, 5' endonuclease activity). Preferably, the polymerase may be selected from any of the following: (a) A polymerase with 5' nuclease activity and polymerization activity; (b) A polymerase having 5' nuclease activity but lacking polymerization activity (e.g., polymerization activity artificially removed or reduced); or, (c) The polymerase as described in (b) and a combination of polymerases having polymerizing activity; Preferably, the DNA polymerase is obtained from the following eubacteria: *Thermus aquaticus* (Taq), *Thermus thermophiles* (Tth), *Thermus filiformis*, *Thermus flavus*, *Thermococcus literalis*, *Thermus antranildanii*, *Thermus caldophllus*, *Thermus chliarophilus*, *Thermus flavus*, *Thermus igniterrae*, *Thermus lacteus*, *Thermus oshimai*, *Thermus ruber*, *Thermus rubens*, *Thermus scotoductus*, *Thermus silvanus*, *Thermus thermophllus*, *Thermotoga maritima*, *Thermotoga neapolitana*, *Thermosipho africanus*, *Thermococcus litoralis*, *Thermococcus barossi*, *Thermococcus gorgonarius*, *Thermotoga maritima*, *Thermotoga... neapolitana, Thermosiphoafricanus, Pyrococcus woesei, Pyrococcushorikoshii, Pyrococcus abyssi, Pyrodictium occultum, Aquifexpyrophilus and Aquifexaeolieus; Particularly preferred is that the DNA polymerase is Taq polymerase.

10. The method according to any one of claims 1-9, wherein, In step (2), the polymerase with 5' nuclease activity cleaves the first oligonucleotide probe that has hybridized with the target nucleic acid and releases the cleavage product; Preferably, the polymerase with 5' nuclease activity cleaves the 5' end or 5' portion of the mediator sequence or the second target-specific sequence of the first oligonucleotide probe, and releases a cleavage product containing a portion (5' end portion) of the mediator sequence or the complete mediator sequence; Preferably, the polymerase having 5' nuclease activity cleaves the 5' portion of the second target-specific sequence of the first oligonucleotide probe (e.g., between the first and second nucleotides at the 5' end); Preferably, the cleavage product contains a complete mediator subsequence; Preferably, the cleavage product comprises the complete mediator sequence and the 5' end nucleotide of the second target-specific sequence.

11. The method according to any one of claims 1-10, wherein in step (3), the presence of one or more cleavage products in step (2) is detected to determine whether one or more target nucleic acids are present in the sample; Preferably, the cleavage products are subjected to melting curve analysis, qPCR-based detection, and / or digital PCR-based detection to determine whether one or more target nucleic acids are present in the sample.

12. The method according to any one of claims 1-11, wherein, The first oligonucleotide probe carries a detectable label; Preferably, the 5' end or 5' portion of the first oligonucleotide probe has a detectable label; Preferably, the detectable marker is selected from: dyes, radioactive markers (e.g., 32 P), binding moieties (e.g., biotin), haptens (e.g., digitoxin), luminescent, phosphorescent or fluorescent moieties (e.g., reporter groups), fluorescent dyes, or any combination thereof; Preferably, the first oligonucleotide probe is labeled with a reporter group and a quencher group, wherein the reporter group is capable of emitting a signal, and the quencher group is capable of absorbing or quenching the signal emitted by the reporter group; and the signal emitted by the first oligonucleotide probe after being cleaved by the polymerase is different from the signal emitted before being cleaved by the polymerase. Preferably, in step (3), the presence of one or more target nucleic acids in the sample is determined by detecting whether the reporter group emits a signal after the first oligonucleotide probe is cleaved by polymerase, thereby detecting whether one or more cleavage products are generated in step (2).

13. The method according to any one of claims 1-12, wherein, In step (3), at least one second oligonucleotide probe is also provided, and the cleavage product of step (2) is contacted with the second oligonucleotide probe under conditions that allow nucleic acid hybridization; Wherein, at least a portion of the second oligonucleotide probe is complementary to all or part of the mediator sequence, and the second oligonucleotide probe carries a detectable label; Preferably, the detectable marker is selected from: dyes, radioactive markers (e.g., 32 P), binding moieties (e.g., biotin), haptens (e.g., digitoxin), luminescent, phosphorescent or fluorescent moieties (e.g., reporter groups), fluorescent dyes, or any combination thereof; Preferably, the second oligonucleotide probe is labeled with a reporter group and a quencher group, wherein the reporter group is capable of emitting a signal, and the quencher group is capable of absorbing or quenching the signal emitted by the reporter group; and the signal emitted by the second oligonucleotide probe after being cleaved by polymerase is different from the signal emitted before being cleaved by polymerase; or, the signal emitted by the second oligonucleotide probe when hybridizing with its complementary sequence is different from the signal emitted when it does not hybridize with its complementary sequence.

14. The method of claim 13, wherein, The signal emitted by the second oligonucleotide probe after being cleaved by polymerase is different from the signal emitted before being cleaved by polymerase; for example, the second oligonucleotide probe generates a detectable signal after being cleaved by polymerase; and, a portion of the sequence of the second oligonucleotide probe can fold itself and form a partially overlapping sequence with the cleavage product. Preferably, the second oligonucleotide probe comprises a complementary sequence and a folded sequence, wherein the complementary sequence is complementary to the cleavage product or a portion thereof, and the folded sequence is capable of forming a partially overlapping sequence with the nucleotide at the 3' end of the cleavage product; For example, the second oligonucleotide probe comprises a first folded sequence, a second sequence, a third folded sequence, and a complementary sequence from 5' to 3', wherein the complementary sequence is complementary to the cleavage product or a portion thereof, and at least a portion of the first folded sequence and at least a portion of the third folded sequence are complementary; Preferably, the complementary sequence is complementary to the mediator sequence contained in the cleavage product, and the complementary sequence is either complementary or non-complementary to the nucleotide at the 5' end of the second target-specific sequence contained in the cleavage product.

15. The method of claim 13, wherein the signal emitted by the second oligonucleotide probe upon hybridization with its complementary sequence differs from the signal emitted when it does not hybridize with its complementary sequence; for example, the second oligonucleotide probe generates a detectable signal upon hybridization with its complementary sequence; wherein, The second oligonucleotide probe comprises, from the 3' to the 5' direction, a template sequence and a capture sequence; Preferably, the second oligonucleotide probe is capable of forming a hairpin structure with a stem and a loop; Preferably, when the second oligonucleotide probe forms a hairpin structure, the template sequence is used to form the stem of the hairpin structure; Preferably, when the second oligonucleotide probe forms a hairpin structure, the capture sequence is contained within the loop of the hairpin structure; Preferably, the capture sequence is complementary to the media sub-sequence or a portion thereof, or the template sequence is complementary to the media sub-sequence or a portion thereof.

16. The method of claim 15, wherein, The capture sequence is complementary to the media sub-sequence or a portion thereof; Preferably, the capture sequence is complementary to the cleavage product or a portion thereof; Preferably, under conditions that allow the polymerase to perform an extension reaction, the polymerase uses the capture sequence as a template to extend the cleavage product hybridized to the capture sequence, thereby forming a double strand.

17. The method of claim 15, wherein, The template sequence is complementary to the mediating subsequence or a portion thereof; Preferably, the template sequence is complementary to the cleavage product or a portion thereof; Preferably, under conditions that allow the polymerase to perform an extension reaction, the polymerase uses the template sequence as a template to extend the cleavage product hybridized to the template sequence, thereby forming a double strand.

18. The method according to any one of claims 1-17, wherein, In step (1), one or more components (e.g., enzymes, primers or primer pairs for amplifying the target nucleic acid) are also provided, and the one or more components are brought into contact with the target nucleic acid under conditions that allow nucleic acid amplification. Preferably, in step (1), for each target nucleic acid to be detected, at least one downstream primer is also provided; wherein the downstream primer contains a sequence complementary to the target nucleic acid sequence; and, when hybridizing with the target nucleic acid sequence, the downstream primer is located downstream of the second target-specific sequence or has a sequence that at least partially overlaps with the second target-specific sequence; Preferably, an isothermal amplification method is used to amplify the target nucleic acid; Preferably, the isothermal amplification is selected from loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), cross-primer amplification (CPA), nucleic acid sequence-based amplification (NASBA), nickase amplification reaction (NARE), helicase-dependent amplification (HAD), or any combination thereof; Preferably, in step (1), one or more enzymes (e.g., enzymes with chain displacement activity (e.g., BST enzymes), polymerases) are also provided; then, under conditions that allow nucleic acid hybridization, the target nucleic acid is contacted with the provided upstream primer, downstream primer, guide probe, first oligonucleotide probe and the enzyme; Preferably, after hybridization with the target nucleic acid, the downstream primer is located at the distal downstream end of the second target-specific sequence, or located adjacent to the downstream of the second target-specific sequence, or has a sequence that partially overlaps with the second target-specific sequence (e.g., the 3' portion of the second target-specific sequence partially overlaps with the 5' portion of the downstream primer), or has a sequence that completely overlaps with the second target-specific sequence, or completely contains the second target-specific sequence. Preferably, in step (1), under conditions that allow nucleic acid amplification, the target nucleic acid is mixed with the upstream primer, downstream primer, and enzyme; then, under conditions that allow nucleic acid hybridization, the guide probe and the first oligonucleotide probe are mixed with the amplification product; or, in step (1), under conditions that allow nucleic acid amplification and hybridization, the target nucleic acid is mixed with the upstream primer, downstream primer, and enzyme, as well as the guide probe and the first oligonucleotide probe.

19. The method according to any one of claims 1-18, wherein in step (1), at least two target nucleic acids to be detected are provided, the target nucleic acids comprising a first region and a second region, wherein the first region is located downstream of the second region; and the second regions of the different target nucleic acids have at least one nucleotide difference; in, For each target nucleic acid to be detected, the following are provided: At least one guide probe includes a first target-specific sequence comprising a sequence at least partially complementary to a first region of a target nucleic acid; optionally, the guide probe further includes a second sequence downstream of the first target-specific sequence; and At least one first oligonucleotide probe includes a mediator sequence and a second target-specific sequence in the 5' to 3' direction, the mediator sequence including a sequence that is not complementary to the target nucleic acid; and the second target-specific sequence including a sequence that is at least partially complementary to a second region of the target nucleic acid; Furthermore, under conditions that allow nucleic acid hybridization, the target nucleic acid is contacted with a guide probe and a first oligonucleotide probe; Preferably, the nucleotide at the 5' end of the second target-specific sequence is complementary to the second region of the target nucleic acid; preferably, the regions where the guide probe and the first oligonucleotide probe hybridize with the target nucleic acid are close (e.g., adjacent, adjacent to, or partially overlapping each other); Preferably, all guide probes are the same or different; Preferably, for each target nucleic acid to be detected, at least one upstream primer is also provided; wherein the upstream primer contains a sequence complementary to the target nucleic acid; and, when hybridizing with the target nucleic acid, the upstream primer is located upstream of the first target-specific sequence or has a sequence that at least partially overlaps with the first target-specific sequence.

20. The method of claim 19, wherein, All first oligonucleotide probes carry different detectable labels; for example, all first oligonucleotide probes carry different reporter groups. Preferably, the mediator sequences of all first oligonucleotide probes are the same or different; Preferably, the second target-specific sequences of all the first oligonucleotide probes are different from each other; Preferably, the 5' end or 5' portion of the first oligonucleotide probe has different detectable markers; Preferably, the detectable marker is selected from: dyes, radioactive markers (e.g., 32 P), binding moieties (e.g., biotin), haptens (e.g., digitoxin), luminescent, phosphorescent, or fluorescent moieties (e.g., reporter groups), fluorescent dyes, or any combination thereof.

21. The method of claim 18 or 19, wherein, The mediator sequences of all first oligonucleotide probes are different from each other; Preferably, in step (3), for each cleavage product, at least one second oligonucleotide probe is provided, the second oligonucleotide probe being able to form a partially overlapping sequence with a portion of the cleavage product (e.g., the nucleotide at the 3' end); the signal emitted by the second oligonucleotide probe after being cleaved by polymerase is different from the signal emitted before being cleaved by polymerase; and different second oligonucleotide probes emit different signals. And under conditions that allow nucleic acid hybridization, the cleavage product of step (2) is contacted with the second oligonucleotide probe. Preferably, in step (3), the cleavage product is subjected to melting curve analysis; and based on the results of the melting curve analysis, it is determined whether the multiple target nucleic acid sequences exist in the sample.

22. The method of claim 18 or 19, wherein, In step (3), at least one second oligonucleotide probe is also provided, the second oligonucleotide probe comprising, from the 3' to 5' direction, a template sequence, and one or more capture sequences complementary to one or more cleavage products or portions thereof (e.g., the second oligonucleotide probe comprising, from the 3' to 5' direction, a template sequence, a first capture sequence complementary to the first cleavage product or portions thereof, and a second capture sequence complementary to the second cleavage product or portions thereof); and, The second oligonucleotide probe is labeled with a reporter group and a quencher group, wherein the reporter group is capable of emitting a signal, and the quencher group is capable of absorbing or quenching the signal emitted by the reporter group; and the signal emitted by the detection probe when hybridizing with its complementary sequence is different from the signal emitted when it does not hybridize with its complementary sequence. And under conditions that allow nucleic acid hybridization, the cleavage product of step (2) is contacted with the second oligonucleotide probe; Preferably, in step (3), the cleavage product is subjected to melting curve analysis; and based on the results of the melting curve analysis, it is determined whether the multiple target nucleic acid sequences exist in the sample.

23. The method of claim 19, wherein, The mediator sequences of all first oligonucleotide probes are different from each other; Preferably, in step (3), for each cleavage product, at least one second oligonucleotide probe is provided, wherein the second oligonucleotide probe comprises, from the 3' to 5' direction, a template sequence complementary to the cleavage product or a portion thereof, and a capture sequence; Furthermore, the signal emitted by the second oligonucleotide probe when it hybridizes with its complementary sequence is different from the signal emitted when it does not hybridize with its complementary sequence; and all second oligonucleotide probes emit different signals. And under conditions that allow nucleic acid hybridization, the cleavage product of step (2) is contacted with the second oligonucleotide probe.

24. The method according to any one of claims 1-23, wherein, The first oligonucleotide probe and the second oligonucleotide probe each independently have one or more features selected from the following: (1) The first oligonucleotide probe and the second oligonucleotide probe each independently comprise or consist of naturally occurring nucleotides (e.g., deoxyribonucleotides or ribonucleotides), modified nucleotides, non-natural nucleotides (e.g., peptide nucleic acids (PNAs) or locked nucleic acids), or any combination thereof; (2) The lengths of the first oligonucleotide probe and the second oligonucleotide probe are each independently 11-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt, 80-90nt, 90-100nt, 100-200nt, 200-300nt, 300-400nt, 400-500nt, 500-600nt, 600-700nt, 700-800nt, 800-900nt or 900-1000nt; (3) The first oligonucleotide probe and the second oligonucleotide probe each independently have a 3'-OH end; or, their 3'-ends are blocked; for example, by adding a chemical moiety (e.g., biotin or alkyl) to the 3'-OH of the last nucleotide of the probe, by removing the 3'-OH of the last nucleotide of the probe, or by replacing the last nucleotide with a dideoxynucleotide, thereby blocking the 3'-end of the detection probe. (4) The first oligonucleotide probe and the second oligonucleotide probe are each independently self-quenching probes; for example, the probe is labeled with a reporter group at its 5' end or upstream and with a quenching group at its 3' end or downstream, or with a reporter group at its 3' end or downstream and with a quenching group at its 5' end or upstream. Preferably, the reporter group and the quencher group are spaced 10-80 nt apart or longer; Preferably, the reporter group is a fluorescent group (e.g., ALEX-350, FAM, VIC, TET, CAL Fluor Gold540, JOE, HEX, CAL Fluor Orange 560, TAMRA, CAL Fluor Red 590, ROX, CAL Fluor Red610, TEXAS RED, CAL Fluor Red 635, Quasar 670, CY3, CY5, CY5.5, Quasar 705); and the quencher group is a molecule or group capable of absorbing / quenching the fluorescence (e.g., DABCYL, BHQ (e.g., BHQ-1 or BHQ-2), ECLIPSE, and / or TAMRA); (5) The length of the second target-specific sequence in the first oligonucleotide probe is 10-140 nt, for example, 10-20 nt, 20-30 nt, 30-40 nt, 40-50 nt, 50-60 nt, 60-70 nt, 70-80 nt, 80-90 nt, 90-100 nt, 100-110 nt, 110-120 nt, 120-130 nt, or 130-140 nt; and (6) The length of the mediator sequence in the first oligonucleotide probe can be 5-140nt, for example 5-10nt, 10-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt, 80-90nt, 90-100nt, 100-110nt, 110-120nt, 120-130nt, 130-140nt; (7) The first oligonucleotide probe is linear or has a hairpin structure; preferably, the hairpin structure can be naturally formed or can be achieved by artificially adding additional bases.

25. The method according to any one of claims 1-24, wherein, The guide probe has one or more features selected from the following: (1) The guide probe comprises or is composed of naturally occurring nucleotides (e.g., deoxyribonucleotides or ribonucleotides), modified nucleotides, non-natural nucleotides (e.g., peptide nucleic acids (PNAs) or locked nucleic acids), or any combination thereof; (2) The length of the guide probe is 15-150nt, for example 15-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt, 80-90nt, 90-100nt, 100-110nt, 110-120nt, 120-130nt, 130-140nt, 140-150nt; (3) The length of the first target-specific sequence in the guide probe is 10-140 nt, for example 10-20 nt, 20-30 nt, 30-40 nt, 40-50 nt, 50-60 nt, 60-70 nt, 70-80 nt, 80-90 nt, 90-100 nt, 100-110 nt, 110-120 nt, 120-130 nt, 130-140 nt; and (4) The guide probe is resistant to nuclease activity (e.g., 5' nuclease activity, such as 5' to 3' exonuclease activity, 5' to 3' endonuclease activity).

26. The method of any one of claims 1-25, wherein, The method has one or more features selected from the following: (1) The upstream primer comprises or consists of naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof; (2) The upstream primer is 15-150nt in length, for example 15-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt, 80-90nt, 90-100nt, 100-110nt, 110-120nt, 120-130nt, 130-140nt, 140-150nt; (3) After hybridization with the target nucleic acid, the upstream primer is located at the distal upstream end of the guide probe, or located adjacent to the upstream of the guide probe, or has a partially overlapping sequence with the guide probe (for example, the 3' part of the upstream primer has a partially overlapping sequence with the 5' part of the guide probe), or has a completely overlapping sequence with the guide probe, or completely contains the sequence of the guide probe. (4) The upstream primer is a primer specific to the target nucleic acid; (5) The downstream primer comprises or consists of naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof; and (6) The length of the downstream primer is 15-150nt, for example 15-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt, 80-90nt, 90-100nt, 100-110nt, 110-120nt, 120-130nt, 130-140nt, 140-150nt.

27. A composition comprising: a polymerase having 5' nuclease activity, and at least one guide probe and at least one first oligonucleotide probe for each target nucleic acid to be detected; wherein, The guide probe includes a first target-specific sequence comprising a sequence at least partially complementary to a first region of a target nucleic acid; optionally, the guide probe further includes a second sequence downstream of the first target-specific sequence; and The first oligonucleotide probe includes a mediator sequence and a second target-specific sequence from the 5' to 3' direction, the mediator sequence comprising a sequence that is not complementary to the target nucleic acid; and the second target-specific sequence comprising a sequence that is at least partially complementary to a second region of the target nucleic acid; The target nucleic acid includes a first region and a second region, and the first region is located downstream of the second region; Preferably, the nucleotide at the 5' end of the second target-specific sequence is complementary to the second region of the target nucleic acid; preferably, the regions where the guide probe and the first oligonucleotide probe hybridize with the target nucleic acid are close (e.g., adjacent, adjacent to, or partially overlapping).

28. The composition of claim 27, wherein, The guide probe does not contain a second sequence; Preferably, the first and second regions of the target nucleic acid are adjacent to each other; Preferably, when hybridizing with the target nucleic acid, the first target-specific sequence hybridizes in region A of the target nucleic acid, and the second target-specific sequence hybridizes in region B of the target nucleic acid; and, in the target nucleic acid, region A is located downstream of region B and adjacent to region B; Preferably, the interval between region A and region B is 5, 4, 3, 2, 1, or 0 nucleotides; preferably, the interval between region A and region B is 2, 1, or 0 nucleotides. Preferably, when the guide probe does not contain a second sequence, the nucleotides of the 3' portion (e.g., the 3' end) of the first target-specific sequence may be complementary to or not complementary to the target nucleic acid; Preferably, when the guide probe does not contain a second sequence, the first target-specific sequence has a 3'-OH end, or its 3' end is closed.

29. The composition of claim 28, wherein, The 3' end of the first target-specific sequence is closed; Preferably, the 3' end of the guide probe is blocked by modifying the 3'-OH group of the nucleotide at the 3' end of the first target-specific sequence (e.g., by modifying an amino group or a phosphate group); or, the 3' end of the guide probe is blocked by adding a chemical moiety (e.g., biotin, alkyl, or a quencher group) to the 3'-OH group of the nucleotide at the 3' end of the first target-specific sequence; or, the 3' end of the guide probe is blocked by removing the 3'-OH group of the nucleotide at the 3' end of the first target-specific sequence; or, the 3' end of the guide probe is blocked by replacing the nucleotide at the 3' end of the first target-specific sequence with a dideoxynucleotide. Preferably, when the guide probe does not contain a second sequence, the 3' end of the guide probe is blocked by modifying the 3'-OH of the nucleotide at the 3' end of the first target-specific sequence with an amino group; Preferably, when the guide probe does not contain a second sequence, the region A and the region B are separated by 0 nucleotides; Preferably, when the guide probe does not contain a second sequence, the nucleotide at the 3' end of the first target-specific sequence is complementary to the target nucleic acid; Preferably, when the guide probe does not contain a second sequence, the nucleotide at the 3' end of the first target-specific sequence is complementary to the target nucleic acid, and the 3' end is closed (e.g., the nucleotide at the 3' end has an amino group modification); and the region A is separated from the region B by 0 nucleotides.

30. The composition of claim 28, wherein, The first target-specific sequence has a 3'-OH terminus; Preferably, when the guide probe does not contain a second sequence, the nucleotide at the 3' end of the first target-specific sequence is not complementary to the target nucleic acid; Preferably, when the guide probe does not contain a second sequence, one or more nucleotides in the 3' portion of the first target-specific sequence are not complementary to the target nucleic acid; preferably, when the guide probe does not contain a second sequence, multiple consecutive nucleotides at the 3' end of the first target-specific sequence are not complementary to the target nucleic acid.

31. The composition of claim 27, wherein, The guide probe also contains a second sequence downstream of the first target-specific sequence; Preferably, the first and second regions of the target nucleic acid are adjacent to each other; Preferably, when hybridizing with the target nucleic acid, the first target-specific sequence hybridizes in region A of the target nucleic acid, and the second target-specific sequence hybridizes in region B of the target nucleic acid; and, in the target nucleic acid, region A and region B are adjacent (e.g., separated by 0 nucleotides); Preferably, when hybridizing with the target nucleic acid, the second sequence is adjacent to the first target-specific sequence, and the length of the second sequence is 5, 4, 3, 2, or 1 nucleotide; preferably, the length of the second sequence is 3, 2, or 1 nucleotide. Preferably, when the guide probe further includes a second sequence downstream of the first target-specific sequence, the nucleotides of the 3' portion (e.g., the 3' end) of the second sequence may be complementary to or not complementary to the target nucleic acid; Preferably, when the guide probe further includes a second sequence downstream of the first target-specific sequence, the second sequence has a 3'-OH end, or its 3' end is closed; Preferably, when hybridizing with the target nucleic acid, the guide probe (e.g., the second sequence of the guide probe), the first oligonucleotide probe (e.g., the second target-specific sequence of the first oligonucleotide probe), and the target nucleic acid have partially overlapping sequences.

32. The composition of claim 31, wherein, The 3' end of the second sequence is closed; Preferably, the 3' end of the guide probe is blocked by modifying the 3'-OH of the nucleotide at the 3' end of the second sequence (e.g., by modifying an amino group or a phosphate group); or, the 3' end of the guide probe is blocked by adding a chemical moiety (e.g., biotin, alkyl, or a quencher group) to the 3'-OH of the nucleotide at the 3' end of the second sequence; or, the 3' end of the guide probe is blocked by removing the 3'-OH of the nucleotide at the 3' end of the second sequence; or, the 3' end of the guide probe is blocked by replacing the nucleotide at the 3' end of the second sequence with a dideoxynucleotide. Preferably, when the guide probe contains a second sequence, the 3' end of the guide probe is blocked by modifying the 3'-OH of the nucleotide at the 3' end of the second sequence with an amino group; Preferably, when the guide probe hybridizes with the target nucleic acid, the second sequence is adjacent to the first target-specific sequence, and the length of the second sequence is 3, 2, or 1 nucleotide; Preferably, when the guide probe contains a second sequence, the nucleotide at the 3' end of the second sequence is complementary to the target nucleic acid.

33. The composition of claim 31, wherein, The second sequence has a 3'-OH terminus; Preferably, when the guide probe hybridizes with the target nucleic acid, the second sequence is adjacent to the first target-specific sequence, and the length of the second sequence is 3, 2, or 1 nucleotide; Preferably, when the guide probe contains a second sequence, the nucleotide at the 3' end of the second sequence is not complementary to the target nucleic acid; Preferably, one or more nucleotides in the 3' portion of the second sequence are not complementary to the target nucleic acid; preferably, multiple consecutive nucleotides at the 3' end of the second sequence are not complementary to the target nucleic acid. Preferably, when the guide probe contains a second sequence, the nucleotide at the 3' end of the second sequence is not complementary to the target nucleic acid, and the nucleotide has a 3'-OH end; and, the region A and the region B are separated by 0 nucleotides, and the length of the second sequence is 1 nucleotide.

34. The composition according to any one of claims 27-33, wherein, For each target nucleic acid to be detected, at least one upstream primer is also provided; wherein the upstream primer contains a sequence complementary to the target nucleic acid; and, when hybridizing with the target nucleic acid, the upstream primer is located upstream of the first target-specific sequence or has a sequence that at least partially overlaps with the first target-specific sequence. Preferably, after hybridization with the target nucleic acid, the upstream primer is located at the distal upstream end of the first target-specific sequence, or located adjacent upstream of the first target-specific sequence, or has a partially overlapping sequence with the first target-specific sequence (e.g., the 3' portion of the upstream primer has a partially overlapping sequence with the 5' portion of the first target-specific sequence), or has a completely overlapping sequence with the first target-specific sequence, or completely contains the first target-specific sequence.

35. The composition according to any one of claims 27-34, wherein, The composition is used to detect the presence of one or more target nucleic acids in a sample, or to characterize and / or quantify one or more target nucleic acids, or to distinguish multiple target nucleic acids, or to detect one or more single nucleotide polymorphism (SNP) sites contained in target nucleic acids, or to identify SNP sites with different genotypes in multiple target nucleic acids. Preferably, the target nucleic acid and / or sample are as defined in claim 8; Preferably, the polymerase having 5' nuclease activity is as defined in claim 9; Preferably, the first oligonucleotide probe is defined as in any one of claims 10-12; Preferably, the composition further comprises at least one second oligonucleotide probe, wherein at least a portion of the second oligonucleotide probe is complementary to all or part of the mediator sequence, and the second oligonucleotide probe is labeled with a detectable tag. Preferably, the second oligonucleotide is as defined in any one of claims 13-17; Preferably, the upstream primer is as defined in claim 26; Preferably, the composition further comprises one or more components for amplifying the target nucleic acid (e.g., an enzyme, primers or primer pairs for amplifying the target nucleic acid), and the one or more components are contacted with the target nucleic acid under conditions that allow nucleic acid amplification; Preferably, the composition further comprises at least one downstream primer (e.g., the downstream primer as defined in claim 26).

36. A kit comprising the composition according to any one of claims 26-35; Preferably, the kit further comprises: one or more enzymes (e.g., enzymes with chain displacement activity (e.g., BST enzyme, polymerase); Preferably, the kit further comprises: reagents for performing nucleic acid hybridization, reagents for cleavage of a first oligonucleotide probe, reagents for nucleic acid extension, reagents for nucleic acid amplification, or any combination thereof.

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