Method for detecting genotype of SNP site in target gene by cascade amplification and kit therefor

Abstract

The present invention provides a method and a kit for detecting the genotype of SNP sites in a target gene by cascade amplification. The method includes designing a first primer, a second primer and a mediator probe for a target gene containing a SNP site to be detected, designing an amplification probe according to the mediator sequence of the mediator probe, designing a detection probe according to the single-stranded reporter sequence of the amplification probe, and performing PCR amplification and analysis of the amplification products in a reaction system containing the upstream first primer, the second primer, the mediator probe, the amplification probe, the detection probe, a nucleic acid sample containing the target gene and a DNA polymerase. The method of the present invention is a non-target-dependent detection method. By detecting a melting peak having the melting point of a certain double-stranded body in melting curve analysis, the SNP typing of the target gene corresponding to the double-stranded body can be determined. With the advantages of PCR amplification and two rounds of PCR exponential amplification of the combination of the mediator primer and the amplification probe in the present invention, a large amount of target nucleic acid can be enriched, and the detection sensitivity can be improved.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid detection, and in particular, to a method for detecting the genotype of SNP sites in a target gene by cascade amplification and a kit thereof. Background Art

[0002] Drug resistance mutation refers to the phenomenon that microorganisms (such as bacteria, viruses, fungi, etc.) undergo genetic variation after exposure to antibiotics or other antimicrobial drugs, resulting in a decrease or complete loss of their sensitivity to the drugs. This variation may be caused by the direct action of the drug on the genetic material (such as DNA) of the microorganism, or may be due to the change of the microorganism's own repair mechanism or metabolic pathway. Drug resistance mutation is a common phenomenon in nature and an important mechanism for microorganisms to resist external pressure. However, drug resistance mutation poses a huge challenge to clinical treatment. At present, drug resistance mutation has become a global public health problem and one of the important factors seriously threatening human health and life safety. Therefore, detecting the mutation situation of microbial gene drug resistance mutation sites is of great significance for guiding clinical medication, reducing unnecessary drug use, promoting the realization of precision medicine, and improving the treatment effect.

[0003] On the other hand, DNA variation occurring at a specific single nucleotide in the genome, namely single nucleotide polymorphism (SNP), is one of the most common genetic variation forms in humans. SNPs are closely related to genetic diseases and have important effects on regulating gene expression, determining human phenotypes, and regulating metabolic processes. It explains the heritable inter-individual differences in complex phenotypes and the relationship between genes and diseases, and is related to disease susceptibility, disease pathogenesis, and individual differences in drug response. Therefore, detecting SNPs can promote the diagnosis, prevention, and treatment of early genetic diseases and has a guiding role in clinical diagnosis.

[0004] Currently, the main methods for detecting gene polymorphisms include direct sequencing of PCR products, polymerase chain reaction - restriction fragment length polymorphism (PCR - RFLP), reverse membrane hybridization, solid-phase gene chips, real-time fluorescence quantitative PCR, amplification refractory mutation system PCR, molecular beacon method, and high-resolution melting curve method, etc.

[0005] Among them, the direct sequencing of PCR products has the advantages of intuitive and reliable results, the ability to analyze unknown DNA sequences and mutation sites, a relatively long read sequence ability in one-way reaction, and high accuracy. However, the detection sensitivity of the conventional PCR method used in the direct sequencing of PCR products is low, which limits its clinical application.

[0006] In addition, the TaqMan probe method is also a common detection method in clinical applications. It has the characteristics of simplicity, high sensitivity, and high accuracy. However, this method only uses the fluorescent signal generated by fluorescence changes for detection and analysis in one dimension. One SNP site requires the design of two specific probes with different fluorescent labels at both ends to identify different alleles. For multiple detection, due to the limitation of fluorescence channels, it can only be used for the analysis of a small number of SNP sites, making it difficult to distinguish multiple targets, and it is more difficult to design probes for SNP sites that are closely spaced.

[0007] Multicolor melting curve analysis combines multicolor fluorescence with melting curve analysis, performing detection and analysis in two dimensions: fluorescence changes and Tm values, achieving multiplexed detection within a single channel. This method utilizes the Tm values generated by probe hybridization with a large number of single-stranded DNA strands for melting curve analysis. Therefore, asymmetric PCR is used to adjust the ratio of upstream and downstream primer concentrations to obtain a large number of single-stranded products complementary to the fluorescent probe. This asymmetric amplification is coupled with the DNA melting temperature to form characteristic peaks in the melting curve. This method circumvents the limitations of PCR instrument fluorescence channels in multiplexed detection methods, offering advantages such as high throughput, ease of operation, low cost, and reliable accuracy. However, multicolor melting curve analysis currently faces numerous challenges. Asymmetric amplification is linear, while non-exponential amplification is prone to low amplification yield and sensitivity. Furthermore, the upstream and downstream primer ratios are difficult to optimize, making design challenging. The molecular beacons used in asymmetric melting curves undergo a process of hairpin formation, free single strands, hybridized double strands, and free single strands during the reaction. The fluorescence signal shifts from weak to strong and from strong to weak, which can easily lead to baseline unevenness and inverted peaks.

[0008] For genetic polymorphism detection, the currently widely used asymmetric melting curve method designs mutation sites on the probe and uses the Tm value difference of a fluorescent probe to distinguish between wild type and mutant types. The Tm value difference for the ATGC>GC mutation is easy to distinguish, but for the A>T mutation, the Tm difference of the probe binding is small, and the genotype is difficult to distinguish. In addition, due to the limitations of probe temperature and two peaks in a single channel, a maximum of 2-3 SNP sites can be designed for one fluorescent channel. Summary of the invention

[0009] In order to solve the above technical problems, the present invention provides a method for detecting the genotype of a SNP site in a target gene using cascade amplification, the method comprising the following steps:

[0010] 1. A method for detecting the genotype of a SNP site in a target gene using cascade amplification, characterized in that the method comprises the following steps:

[0011] Step 1: Design the first primer, second primer and intermediate probe for the target gene containing the SNP site to be detected.

[0012] 1.1. The first primer and the second primer specifically bind to the target gene and are used to amplify the target gene containing the SNP site to be detected;

[0013] 1.2. The mediator probe sequentially includes a mediator sequence and a target gene-specific binding sequence from the 5'-end to the 3'-end direction, wherein the mediator sequence cannot bind to the target gene; the target gene-specific binding sequence can specifically and complementarily bind to the target gene, and along the 5'-end to the 3'-end direction, the first base sequence of the target gene-specific binding sequence corresponds to the SNP site of the target gene, and the first base sequence is complementary to the wild-type base or the mutant base of the SNP site of the target gene; the 3'-end of the mediator probe is labeled with a group that blocks extension;

[0014] Step 2: Design an amplification probe according to the mediator sequence of the mediator probe in step one. The amplification probe sequentially includes a single-stranded reporter sequence, a double-stranded auxiliary sequence, and a single-stranded mediator sequence-specific binding sequence. The single-stranded reporter sequence does not bind to the target gene, and the single-stranded reporter sequence does not complementarily pair with other sequences in the amplification probe; the double-stranded structure of the auxiliary sequence remains stable during the PCR amplification process; the mediator sequence-specific binding sequence specifically and complementarily binds to the mediator sequence in the mediator probe, and the double-stranded formed after the binding of the mediator sequence and the mediator sequence-specific binding sequence is exactly flush with the double-stranded of the auxiliary sequence, that is, there is no spacer base between the above two double-strands; the end of the single-stranded mediator sequence-specific binding sequence is labeled with a group that blocks extension;

[0015] Step 3: Design a detection probe according to the single-stranded reporter sequence of the amplification probe. The detection probe sequentially includes a complementary sequence to the single-stranded reporter sequence of the amplification probe, a base complementary to the first base at the 5'-end of the auxiliary sequence, and an extension sequence. For the extension sequence, along the 3'-end to the 5'-end direction, the first base cannot be complementary to the second base of the auxiliary sequence along the 5'-end to the 3'-end direction;

[0016] Step 4: In a reaction system containing the first primer, the second primer, the mediator probe, the amplification probe, the detection probe, a nucleic acid sample containing the target gene, and a DNA polymerase, perform PCR amplification and analysis of the amplification product:

[0017] 4.1. When the first base of the target gene-specific binding sequence is complementary to the wild-type base of the SNP site of the target gene:

[0018] 4.1.1. When the SNP site of the target gene is wild-type, at this time, the first base of the target gene specific binding sequence in the media probe is complementary to the wild-type target gene to form a SNP site base pair. Both the first primer and the second primer are extended for amplification. When extending from the 5'-end to the 3'-end to the last base of the media sequence in the media probe, DNA polymerase will cleave the phosphodiester bond between the SNP site base pair and the first adjacent base pair. The cleaved sequence fragment contains the media sequence and the first base, and this fragment is called the first media primer. This media primer specifically binds to the single-stranded media sequence specific binding sequence of the amplification probe and forms an invasion structure of a single base. After this structure is formed, DNA polymerase will cut off the single-stranded reporter sequence of the amplification probe together with the invaded first base. The cleaved sequence fragment contains the single-stranded reporter sequence together with the invaded first base, and this sequence fragment is called the first reporter primer; the first reporter primer is completely complementary to the detection probe and extends along the extended sequence part of the detection probe to form a first double-stranded product. When only the formation of the first double-stranded product is detected, it can be determined that the SNP site in the nucleic acid sample containing the target gene is wild-type;

[0019] 4.1.2. When the SNP site of the target gene is mutant, at this time, the first base of the target gene specific binding sequence in the media probe is not complementary to the mutant target gene. At this time, neither the first base of the target gene specific binding sequence nor the media sequence is complementary to the target gene. Both the first primer and the second primer are extended for amplification. When extending from the 5'-end to the 3'-end to the first base of the target gene specific binding sequence in the media probe, DNA polymerase will cleave the phosphodiester bond between the first base and the second base that are paired with the target gene in the target gene specific binding sequence. The cleaved sequence fragment contains the media sequence, the first base of the target gene specific binding sequence, and the first base that is paired with the target gene, and this fragment is called the second media primer; the second media primer specifically binds to the amplification probe and forms an invasion structure of two bases. After this structure is formed, DNA polymerase will cut off the reporter sequence of the amplification probe together with the invaded first and second bases, and this sequence fragment is called the second reporter primer; when the second reporter primer is complementary to the detection probe, the last base at the 3'-end of the second reporter primer cannot be complementary to the detection probe, and the second reporter primer cannot extend along the detection probe. Therefore, a second double-stranded product is obtained. When only the formation of the second double-stranded product is detected, it can be determined that the SNP site in the nucleic acid sample containing the target gene is mutant;

[0020] 4.1.3. When the SNP site of the target gene is heterozygous, the first double-stranded product and the second double-stranded product will be generated simultaneously. Therefore, when it is detected that both the first double-stranded product and the second double-stranded product are formed, it can be determined that the SNP site in the nucleic acid sample containing the target gene is heterozygous;

[0021] 4.2. When the first base sequence of the target gene-specific binding sequence is complementary to the missense mutant base of the SNP site of the target gene:

[0022] 4.2.1. If the SNP site of the target gene is wild-type or synonymous mutant, at this time, the first base of the target gene-specific binding sequence in the mediator probe does not bind complementarily to the target gene. At this time, neither the first base of the target gene-specific binding sequence nor the mediator sequence binds complementarily to the target gene. Just like in 4.1.2 above, when only the formation of the second double-stranded product is detected, it can be determined that the SNP site in the nucleic acid sample containing the target gene is wild-type or synonymous mutant;

[0023] 4.2.2. If the SNP site of the target gene is a missense mutant, at this time, the first base of the target gene-specific binding sequence in the mediator probe binds complementarily to the missense mutant target gene to form an SNP site base pair. Just like in 4.1.1 above, when only the formation of the first double-stranded product is detected, it can be determined that the SNP site in the nucleic acid sample containing the target gene is a missense mutant;

[0024] 4.2.3. Just like in 4.1.3 above, when it is detected that both the first double-stranded product and the second double-stranded product are formed, it can be determined that the SNP site in the nucleic acid sample containing the target gene is heterozygous.

[0025] In one embodiment, when there are multiple SNP sites in the target gene, corresponding mediator probes, amplification probes, and detection probes are designed for each SNP site respectively, so that the first double-stranded product and the second double-stranded product corresponding to each SNP site are formed respectively.

[0026] In one embodiment, the method of melting curve is used to detect whether the first double-stranded product and / or the second double-stranded product is formed.

[0027] In one embodiment, a fluorescent group is labeled on the single-stranded reporter sequence of the amplification probe; a quenching group corresponding to the fluorescent group labeled on the single-stranded reporter sequence is labeled on the double-stranded auxiliary sequence of the amplification probe; a quenching group corresponding to the fluorescent group labeled on the single-stranded reporter sequence of the amplification probe is modified on the detection probe, and whether the first double-stranded product and / or the second double-stranded product is formed is detected by means of a melting curve.

[0028] In one embodiment, a fluorescent group and a corresponding quenching group are labeled on the detection probe, and whether the first double-stranded product and / or the second double-stranded product is formed is detected by means of a melting curve.

[0029] In one embodiment, the mediator probe sequence, the amplification probe, and / or the detection probe sequence comprises or consists of naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof.

[0030] In one embodiment, the DNA polymerase includes at least one of Taq DNA polymerase, Tth DNA polymerase, Tfl DNA polymerase, Tfi DNA polymerase, pfu DNA polymerase, KOD DNA polymerase, or Tgo DNA polymerase.

[0031] In one embodiment, a kit for detecting the genotype of an SNP site in a target gene is provided, and the kit includes: a DNA polymerase, a first primer, a second primer, a mediator probe, an amplification probe, and a detection probe designed for the target gene containing the SNP site to be detected.

[0032] a. The first primer and the second primer specifically bind to the target gene and are used for amplifying the target gene containing the SNP site to be detected.

[0033] b. The mediator probe sequentially includes a mediator sequence and a target gene-specific binding sequence from the 5'-end to the 3'-end direction, wherein the mediator sequence cannot bind to the target gene; the target gene-specific binding sequence can specifically and complementarily bind to the target gene, and along the 5'-end to the 3'-end direction, the first base sequence of the target gene-specific binding sequence corresponds to the SNP site of the target gene, and the first base sequence is complementary to the wild-type base or the mutant base of the SNP site of the target gene; a group that blocks extension is labeled at the 3'-end of the mediator probe.

[0034] c. Design amplification probes according to the mediator sequence of the mediator probe. The amplification probes sequentially include a single-stranded reporter sequence, a double-stranded auxiliary sequence, and a single-stranded mediator sequence-specific binding sequence. The single-stranded reporter sequence does not bind to the target gene, and the single-stranded reporter sequence does not complementarily pair with other sequences in the amplification probe. The double-stranded structure of the auxiliary sequence remains stable during the PCR amplification process. The mediator sequence-specific binding sequence specifically and complementarily binds to the mediator sequence in the mediator probe, and the double-stranded formed after the binding of the mediator sequence and the mediator sequence-specific binding sequence is exactly flush with the double-stranded of the auxiliary sequence, that is, there are no spacer bases between the above two double-strands. The end of the single-stranded mediator sequence-specific binding sequence is labeled with a group that blocks extension;

[0035] d. Design detection probes according to the single-stranded reporter sequence of the amplification probe. The detection probes sequentially include a complementary sequence to the single-stranded reporter sequence of the amplification probe, a base complementary to the first base at the 5'-end of the auxiliary sequence, and an extension sequence. In the extension sequence, along the 3'-end to 5'-end direction, the first base cannot be complementary to the second base of the auxiliary sequence along the 5'-end to 3'-end direction.

[0036] Compared with the prior art, the present invention has the following advantages:

[0037] 1. The method of the present invention includes two amplification cycles. The first amplification cycle is PCR amplification. As the PCR amplification reaction proceeds, a continuous supply of mediator primers is generated. The second amplification is the binding of the mediator primers to the amplification probes, cutting and releasing the reporter primers. Subsequently, the reporter primers continuously bind to the detection probes to form double-stranded products. In the second amplification cycle, the mediator primers are not consumed, but act as a catalyst. As the amplification reaction proceeds, they continuously bind to the amplification probes and continuously cut and release the reporter primers. However, the two amplification cycles are interconnected and independent. Since the mediator primers are not consumed, even if the PCR amplification cycle stops, it does not affect the continuous occurrence of the second amplification cycle, thereby realizing the cascade amplification of the detection of the target gene and further improving the detection sensitivity;

[0038] 2. The method of the present invention is a non-target-dependent detection method, that is, the sequences of each reporting primer and detection probe are artificially designed, known or predetermined. Therefore, the first double-stranded product and the second-stranded product can be determined. In the melting curve detection, the melting point (Tm value) of each double-stranded body can be calculated in advance. Thus, by detecting the melting peak with the melting point (Tm value) of a certain double-stranded body in the melting curve analysis, the SNP typing of the target gene corresponding to the double-stranded body can be judged, which solves the problem of the melting curve peak shift and easy misjudgment caused by the easy mutation of the target gene sequence to be detected, and well solves the problem that the melting curve analysis method is not applicable to RNA samples;

[0039] 3. In the currently widely used asymmetric melting curve method, the mutation site is designed on the probe, and the Tm value difference of a fluorescent probe is used to distinguish the wild type and the mutant type. The Tm value difference is easy to distinguish for the mutation of ATGC>GC, but for the mutation of A>T, the Tm difference of probe binding is small, and the genotype is difficult to distinguish. At present, the asymmetric melting curve is widely used in SNP genotyping. In this method, the mutation site is designed on the probe. Since the probe has no selectivity, only one probe is designed for one SNP site to distinguish two genotypes, and the genotype is distinguished by the single-base Tm value of the probe binding to the single strand in the same fluorescence channel. The Tm value difference is easy to distinguish for the mutation of ATGC>GC, but for the mutation of A>T, the Tm difference of probe binding is small, and the genotype is difficult to distinguish. And because of the limitations of the probe temperature and the two peaks of the single channel, at most 2-3 SNP sites can be designed in one fluorescence channel. In the method of the present invention, the first base of the specific binding sequence of the mediator probe and the target gene is designed as the SNP site. Whether the SNP site of the target gene to be detected is complementary to the mediator probe is used to cut and generate two different mediator primers. Due to the difference in the 3'-terminal sequence of different mediator primers, different invaded structures with different base numbers will be formed after binding to the amplification probe. Different invaded structures will generate two different reporting primers. Due to the difference in the 3'-terminal sequence of the two reporting primers, after binding to the detection probe, only one of the reporting primers can extend along the detection probe, while the other reporting primer can only bind to the detection probe but cannot extend. Therefore, the melting point (Tm value) of the double-stranded body can be arbitrarily constructed, realizing the customization of the melting curve peak of the SNP site, thus solving the problem that the Tm values of the wild type and mutant type of the genotype at the same SNP site cannot be distinguished, reducing the design difficulty, and enabling the specific distinction of the A>T base mutation of the detected target site. For different SNP sites, the Tm values are distinguished by controlling the amplicon length and GC content between the reporting primer and the detection probe, and different SNP sites are distinguished by different melting curve Tm values, so the number of detectable SNP sites is increased.

[0040] 4. In the method of the present invention, the design of the double-stranded auxiliary sequence part of the amplification probe ingeniously forms a large steric hindrance, preventing the binding of excessive amplification probes to the detection probe to generate false positive background peaks, and avoiding the problem that the false positive background peaks interfere with the accuracy of interpretation;

[0041] 5. In the method of the present invention, when using the melting curve method to detect the first double-stranded product and the second-stranded product, during the melting curve analysis process, the fluorescence signal fluctuations of the reporting primer and the detection probe are more prominently reflected, obtaining a flatter baseline, reducing the appearance of non-specific peaks, and improving the correct rate and accuracy of the detection method. At the same time, this method is not limited by the number of fluorescence channels detected. Different target genes are distinguished according to the melting point and fluorescence color, increasing the number of target genes detected in a single well and improving the detection throughput. With the method of the present invention, multiple target detections can be performed. The baselines of the melting peaks can all remain flat, the peaks are higher, and there are no non-specific peaks, with higher sensitivity; at the same time, the detection interval range is also wider, and the melting curve detection T m value range is: 40~80°C;

[0042] 6. When using the melting curve method for detection, the method for detecting target nucleic acid in the present invention is a symmetric amplification melting curve method. Compared with the common asymmetric amplification melting curve method (a linear amplification method), this method utilizes the advantage of PCR exponential amplification to enrich a large amount of target nucleic acid and improve the detection sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings described below are only some embodiments recorded in the present application. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0044] Figure 1 Schematic diagram of the amplification probe used in the method of the present invention;

[0045] Figure 2 Schematic diagram of the basic principle of the method of the present invention;

[0046] Figure 3 Melting curve analysis diagram of the MTHFR (677C>T) genotype of CC wild type;

[0047] Figure 4 Melting curve analysis diagram of the MTHFR (677C>T) genotype of TT homozygous mutant;

[0048] Figure 5 Melting curve analysis diagram of the MTHFR (677C>T) genotype of CT heterozygous mutant;

[0049] Figure 6 Melting curve analysis diagram of the AA wild type of the MTHFR (1298A>C) genotype;

[0050] Figure 7 Melting curve analysis diagram of the CC homozygous mutant of the MTHFR (1298A>C) genotype;

[0051] Figure 8 Melting curve analysis diagram of the AC heterozygous mutant of the MTHFR (1298A>C) genotype;

[0052] Figure 9 Melting curve analysis diagram of both SNP sites being mutant;

[0053] Figure 10 Melting curve analysis diagram of both SNP sites being wild type;

[0054] Figure 11 Melting curve analysis diagram of MTHFR (677C>T) being wild type and MTHFR (1298A>C) being mutant;

[0055] Figure 12 Melting curve analysis diagram of MTHFR (677C>T) being wild type and MTHFR (1298A>C) being heterozygous;

[0056] Figure 13 Melting curve analysis diagram of MTHFR (677C>T) being heterozygous and MTHFR (1298A>C) being mutant;

[0057] Figure 14 Melting curve analysis diagram of MTHFR (677C>T) being heterozygous and MTHFR (1298A>C) being heterozygous. Specific implementation manners

[0058] In order to enable those skilled in the art to better understand the technical solutions in this application, the present invention will be further described below in conjunction with embodiments. Obviously, the described embodiments are only a part of the embodiments of this application, rather than all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of this application. In the following embodiments, unless otherwise specified, they are all conventional methods in the art.

[0059] Embodiment 1 Basic principle of the present invention

[0060] Such as Figure 1 And Figure 2As shown in the figure, the present invention provides a method for detecting the genotype of SNP sites in a target gene by cascade amplification. The method comprises the following steps:

[0061] Step 1: Design a first primer, a second primer and a mediator probe for the target gene containing the SNP site to be detected.

[0062] 1.1. The first primer and the second primer specifically bind to the target gene and are used for amplifying the target gene containing the SNP site to be detected.

[0063] 1.2. The mediator probe sequentially includes a mediator sequence and a target gene specific binding sequence from the 5'-end to the 3'-end direction, wherein the mediator sequence cannot bind to the target gene; the target gene specific binding sequence can specifically and complementarily bind to the target gene, and along the 5'-end to the 3'-end direction, the first base sequence of the target gene specific binding sequence corresponds to the SNP site of the target gene, and the first base sequence is complementary to the wild-type base or mutant base of the SNP site of the target gene; a group that blocks extension is labeled at the 3'-end of the mediator probe.

[0064] Step 2: Design an amplification probe according to the mediator sequence of the mediator probe in Step 1. As Figure 1 shown in a, the amplification probe of the present invention can be designed in various different forms. The amplification probe can be composed of one strand, or can be composed of two strands or multiple strands; the amplification probe sequentially includes a single-stranded reporter sequence, a double-stranded auxiliary sequence and a single-stranded mediator sequence specific binding sequence. The single-stranded reporter sequence does not bind to the target gene, and the single-stranded reporter sequence does not complementarily pair with other sequences in the amplification probe; the double-stranded structure of the auxiliary sequence remains stable during the PCR amplification process; the mediator sequence specific binding sequence specifically and complementarily binds to the mediator sequence in the mediator probe, and the double-stranded formed after the binding of the mediator sequence and the mediator sequence specific binding sequence is exactly flush with the double-stranded of the auxiliary sequence, that is, there is no spacer base between the above two double-strands (as Figure 1 shown in b); a group that blocks extension is labeled at the end of the single-stranded mediator sequence specific binding sequence.

[0065] Step 3: Design a detection probe according to the single-stranded reporter sequence of the amplification probe. The detection probe sequentially includes a complementary sequence to the single-stranded reporter sequence of the amplification probe, a base complementary to the first base at the 5'-end of the auxiliary sequence and an extension sequence. For the extension sequence, along the 3'-end to 5'-end direction, the first base cannot be complementary to the second base of the auxiliary sequence along the 5'-end to 3'-end direction.

[0066] Step 4: In the reaction system containing the first primer, the second primer, the mediator probe, the amplification probe, the detection probe, the nucleic acid sample containing the target gene, and DNA polymerase, perform PCR amplification and analysis of the amplification product:

[0067] 4.1. When the first base of the target gene specific binding sequence is complementary to the wild-type base of the SNP site of the target gene:

[0068] 4.1.1. When the SNP site of the target gene is wild-type, at this time, the first base of the target gene specific binding sequence in the mediator probe binds complementarily to the wild-type target gene to form a SNP site base pair. Both the first primer and the second primer are extended for amplification. When extending from the 5' end to the 3' end to the last base of the mediator sequence in the mediator probe, DNA polymerase will cleave the phosphodiester bond between the SNP site base pair and the first adjacent base pair. The cleaved sequence fragment contains the mediator sequence and the first base, and this fragment is called the first mediator primer. This mediator primer specifically binds to the single-stranded mediator sequence specific binding sequence of the amplification probe and forms an invasion structure of a single base (such as Figure 1 c). After the formation of this structure, DNA polymerase cleaves off the single-stranded reporter sequence of the amplification probe together with the invaded first base. The cleaved sequence fragment contains the single-stranded reporter sequence together with the invaded first base, and this sequence fragment is called the first reporter primer; the first reporter primer completely complementary pairs with the detection probe and extends along the extended sequence part of the detection probe to form a first double-stranded product. When only the formation of the first double-stranded product is detected, it can be determined that the SNP site in the nucleic acid sample containing the target gene is wild-type;

[0069] 4.1.2. When the SNP site of the target gene is mutant, at this time, the first base of the target gene specific binding sequence in the media probe does not complementarily bind to the mutant target gene. At this time, neither the first base of the target gene specific binding sequence nor the media sequence complementarily binds to the target gene. Both the first primer and the second primer perform extension amplification. When extending from the 5' end to the 3' end to the first base of the target gene specific binding sequence in the media probe, DNA polymerase will cleave the phosphodiester bond between the first base and the second base that pair with the target gene in the target gene specific binding sequence. The cleaved sequence fragment contains the media sequence, the first base of the target gene specific binding sequence, and the first base that pairs with the target gene, and this fragment is called the second media primer; the second media primer specifically binds to the amplification probe and forms an invasion structure of two bases. After this structure is formed, DNA polymerase will cut off the reporter sequence of the amplification probe together with the invaded first and second bases, and this sequence fragment is called the second reporter primer; when the second reporter primer complementarily binds to the detection probe, the last base at the 3' end of the second reporter primer cannot complementarily bind to the detection probe, and the second reporter primer cannot extend along the detection probe, so a second double-stranded product is obtained. When only the formation of the second double-stranded product is detected, it can be determined that the SNP site in the nucleic acid sample containing the target gene is mutant;

[0070] 4.1.3. When the SNP site of the target gene is heterozygous, at this time, the first double-stranded product and the second double-stranded product will be generated simultaneously. Therefore, when the formation of the first double-stranded product and the second double-stranded product is detected simultaneously, it can be determined that the SNP site in the nucleic acid sample containing the target gene is heterozygous;

[0071] 4.2. When the first base sequence of the target gene specific binding sequence is complementary to the missense mutant base of the SNP site of the target gene:

[0072] 4.2.1. When the SNP site of the target gene is wild-type or synonymous mutant, at this time, the first base of the target gene specific binding sequence in the media probe does not complementarily bind to the target gene. At this time, neither the first base of the target gene specific binding sequence nor the media sequence complementarily binds to the target gene. Just like in 4.1.2 above, when only the formation of the second double-stranded product is detected, it can be determined that the SNP site in the nucleic acid sample containing the target gene is wild-type or synonymous mutant;

[0073] 4.2.2. When the SNP site of the target gene is a missense mutation type, at this time, the first base of the target gene specific binding sequence in the medium probe is complementary to the missense mutation type target gene to form a SNP site base pair. Just like in 4.1.1 above, when only the formation of the first double-stranded product is detected, it can be determined that the SNP site in the nucleic acid sample containing the target gene is of the missense mutation type;

[0074] 4.2.3. Just like in 4.1.3 above, when the formation of both the first double-stranded product and the second double-stranded product is detected, it can be determined that the SNP site in the nucleic acid sample containing the target gene is of the heterozygous type.

[0075] In the method of the present invention, a fluorescent group is labeled on the single-stranded reporter sequence of the amplification probe; a quenching group corresponding to the fluorescent group labeled on the single-stranded reporter sequence is labeled on the double-stranded auxiliary sequence of the amplification probe; a quenching group corresponding to the fluorescent group labeled on the single-stranded reporter sequence of the amplification probe is modified on the detection probe, and whether the first double-stranded product and / or the second double-stranded product is formed is detected by the method of melting curve.

[0076] In the method of the present invention, a fluorescent group and a corresponding quenching group are labeled on the detection probe, and whether the first double-stranded product and / or the second double-stranded product is formed is detected by the method of melting curve.

[0077] In the method of the present invention, in addition to DNA polymerase, the amplification reaction system may also contain endonuclease and / or exonuclease.

[0078] In the method of the present invention, the invading base can be complementary to the sense strand or the antisense strand of the double-stranded auxiliary sequence of the amplification probe, or it can not be complementary to any strand of the double-stranded auxiliary sequence of the amplification probe.

[0079] In the present invention, since the base sequence and length of the detection probe can be designed in advance, the first double-stranded product obtained by the extension of the first reporter primer and the detection probe has a predictable length and structure. Correspondingly, the first double-stranded product has a predictable melting point (T m1 value). Similarly, the second double-stranded product obtained by the binding of the second reporter primer and the detection probe also has a predictable length and structure. Correspondingly, the second double-stranded product has a predictable melting point (T m2 value). And because the first double-stranded product has a longer base sequence and structure than the second double-stranded product, so T m1 > T m2 , perform melting curve analysis and detection on the double-stranded product obtained in step four. By detecting the peak position of the melting curve of the double-stranded product, the corresponding genotype can be determined.

[0080] In the present invention, the fluorescent group includes various commonly used fluorescent markers at present, but is not limited to these fluorescent markers, such as Pacific Blue, Oregon Green, Bodipy FL-X, FAM, VIC, TET, Bodipy R6G-X, JOE, HEX, Cy3, Cy3B, Rhodamine Red, TAMRA, Texas Red-X, ROX, Cy3.5, Cy5, etc.; the quenching group includes various quenching agents commonly used at present, but is not limited to these quenching agents, such as Dabcyl, Eclipse, BHQ-1, BHQ-2, QYS-7, etc.

[0081] Example 2 Genotyping of MTHFR C677T Locus

[0082] In this example, taking the detection of MTHFR C677T locus as an example, the MTHFR gene was detected by the method of the present invention. The specific method includes the following steps.

[0083] I. Primer and Probe Sequence Information

[0084] According to the target nucleic acid site to be detected, a first primer, a second primer, a mediator probe, an amplification probe and a detection probe were designed. Among them, the first base sequence of the target gene specific binding sequence was used to design the mediator probe complementary to the missense mutant base of the SNP site of the target gene. The sequence information is shown in Table 1 below.

[0085] Table 1

[0086]

[0087] Note: The bold part of the mediator probe is the mediator sequence, the italic part is the SNP site, and the lowercase letters are the target gene specific sequences; the bold part of the amplification probe is the reporter sequence, the part with capital letters and underlines is the double-stranded auxiliary sequence, and the italic part is the mediator sequence specific binding sequence.

[0088] II. PCR Amplification Reaction System and Program

[0089] The present invention uses a 25 μL PCR reaction system for PCR amplification and melting curve analysis. The PCR reaction system includes: 1× PCR buffer, 2.5 U BioZues ® HS Multiplex Taq DNA Polymerase, 0.1 mM dNTPs, 40 nM first primer, 40 nM second primer, 20 nM mediator probe, 100 nM amplification probe, 100 nM detection probe 1, 5 μL nucleic acid template.

[0090] The fluorescence PCR reaction program of the present invention is pre-denaturation at 95°C for 5 min; denaturation at 95°C for 15 s, annealing and extension at 60°C for 45 s while collecting fluorescence, repeating 45 cycles; the melting curve program is 95°C for 2 min, 40°C for 5 min, and 40 - 80°C for melting curve analysis, detecting the fluorescence signal every 0.04°C. The instrument used in this example is the SLAN 96 Real-Time Fluorescence PCR Instrument (Shanghai Hongshi Medical Technology Co., Ltd.).

[0091] III. Melting Curve Analysis

[0092] The results of the melting curve analysis are shown in Table 2 below.

[0093] Table 2

[0094]

[0095] For the MTHFR C677T mutation site, the VIC channel peaks at a T m value of 54.5 ± 1°C and is judged as wild type, as shown in Figure 3 ; the VIC channel peaks at a T m value of 67.1 ± 1°C and is judged as homozygous mutant, as shown in Figure 4 ; the VIC channel peaks simultaneously at T m values of 54.6 ± 1°C and 67.3 ± 1°C and is judged as heterozygous, as shown in Figure 5 .

[0096] Example 3 Genotyping of the MTHFR A1298C Site

[0097] Taking the detection of the MTHFR A1298C mutation site as an example, the method of the present invention is used to detect the mutation of the MTHFR gene, and the specific method includes the following steps.

[0098] I. Primer and Probe Sequence Information

[0099] According to the target nucleic acid site to be detected, a first primer, a second primer, a mediator probe, an amplification probe, and a detection probe are designed. Among them, the first base sequence of the target gene-specific binding sequence is designed as a mediator probe complementary to the missense mutant base of the SNP site of the target gene, and the sequence information is shown in Table 3 below.

[0100] Table 3

[0101]

[0102] Note: For the media probe, the bold part is the media sequence, the italic part is the SNP site, and the lowercase letter part is the target gene specific sequence; for the amplification probe, the bold part is the reporter sequence, the underlined part in capital letters is the double-stranded auxiliary sequence, and the italic part is the media sequence specific binding sequence.

[0103] II. PCR Amplification Reaction System and Procedure

[0104] The present invention uses a 25 μL PCR reaction system for PCR amplification and melting curve analysis. The PCR reaction system includes: 1× PCR buffer, 2.5 U BioZues ® HS Multiplex Taq DNA Polymerase, 0.1 mM dNTPs, 40 nM first primer, 40 nM second primer, 20 nM media probe, 100 nM amplification probe, 100 nM detection probe 2, and 5 μL nucleic acid template.

[0105] The fluorescence PCR reaction procedure of the present invention is: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 15 s, annealing and extension at 60°C for 45 s while collecting fluorescence, repeating 45 cycles; the melting curve procedure is 95°C for 2 min, 40°C for 5 min, and 40 - 80°C for melting curve analysis, detecting the fluorescence signal every 0.04°C. The instrument used in this example is the SLAN 96 Real-Time Fluorescence PCR Instrument (Shanghai Hongshi Medical Technology Co., Ltd.).

[0106] III. Melting Curve Analysis

[0107] The results of the melting curve analysis are shown in Table 4 below.

[0108] Table 4

[0109]

[0110] For the MTHFR A1298C mutation site, the ROX channel peaks at the T m value of 48.0 ± 1°C and is judged as wild type, as Figure 6 ; the ROX channel peaks at the T m value of 65.3 ± 1°C and is judged as homozygous mutant, as Figure 7 ; the ROX channel peaks simultaneously at the T m values of 48.1 ± 1°C and 65.2 ± 1°C and is judged as heterozygous, as Figure 8 .

[0111] Example 3 Genotyping of MTHFR C677T and A1298C Double Loci

[0112] In this embodiment, taking the detection of the double mutation sites of MTHFR C677T and MTHFR A1298C as an example, the method of the present invention is used to detect the mutations of the MTHFR gene. The specific method includes the following steps:

[0113] I. Primer and probe sequence information

[0114] Design the first primer, the second primer, the mediator probe, the amplification probe and the detection probe according to the target nucleic acid site to be detected respectively. Among them, the first base sequence of the specific binding sequence of the target gene is designed as the mediator probe complementary to the missense mutation type base of the SNP site of the target gene. The sequence information is shown in Table 5 below.

[0115] Table 5

[0116]

[0117] Note: The bold part of the mediator probe is the mediator sequence, the italic part is the SNP site, and the lowercase letter part is the target gene specific sequence; the bold part of the amplification probe is the reporter sequence, the part of uppercase letters with underline is the double-stranded auxiliary sequence, and the italic part is the specific binding sequence of the mediator sequence.

[0118] II. PCR amplification reaction system and procedure

[0119] The present invention uses a 25 μL PCR reaction system for PCR amplification and melting curve analysis. The PCR reaction system includes: 1× PCR buffer, 5 U BioZues ® HS Multiplex Taq DNA Polymerase, 0.2 mM dNTPs, 40 nM the first primer, 40 nM the second primer, 20 nM the mediator probe, 100 nM the amplification probe, 100 nM detection probe 1 and 100 nM detection probe 2, 5 μL nucleic acid template.

[0120] The fluorescence PCR reaction procedure of the present invention is pre-denaturation at 95°C for 5 min; denaturation at 95°C for 15 s, annealing and extension at 60°C for 45 s and collecting fluorescence at the same time, repeating 45 cycles; the melting curve procedure is 95°C for 2 min, 40°C for 5 min, and 40 - 80°C is for melting curve analysis, detecting the fluorescence signal every 0.04°C. The instrument used in this embodiment is the SLAN 96 real-time fluorescence PCR instrument (Shanghai Hongshi Medical Technology Co., Ltd.).

[0121] For the one-tube detection of the double sites of MTHFR C677T and MTHFR A1298C, the melting curve results of the random template are as attached Figures 9 to 14 ;

[0122] a. If the VIC channel peaks only at 67.3 ± 1 °C and the ROX channel peaks only at 65.2 ± 1 °C, then both loci are homozygous mutant ( Figure 9 );

[0123] b. If the VIC channel peaks only at 54.6 ± 1 °C and the ROX channel peaks only at 48.1 ± 1 °C, then both loci are wild type ( Figure 10 );

[0124] c. If the VIC channel peaks only at 54.6 ± 1 °C and the ROX channel peaks only at 65.2 ± 1 °C, then C677T is wild type and the A1298C locus is homozygous mutant ( Figure 11 );

[0125] d. If the VIC channel peaks only at 54.6 ± 1 °C and the ROX channel peaks at both 48.1 ± 1 °C and 65.2 ± 1 °C, then C677T is wild type and the A1298C locus is heterozygous mutant ( Figure 12 );

[0126] e. If the VIC channel peaks at both 54.6 ± 1 °C and 67.3 ± 1 °C and the ROX channel peaks only at 65.2 ± 1 °C, then C677T is heterozygous mutant and the A1298C locus is homozygous mutant ( Figure 13 );

[0127] f. If the VIC channel peaks at both 54.6 ± 1 °C and 67.3 ± 1 °C and the ROX channel peaks at both 48.1 ± 1 °C and 65.2 ± 1 °C, then both the C677T and A1298C loci are heterozygous mutants ( Figure 14 ).

[0128] III. Melting Curve Analysis

[0129] The results of the melting curve analysis are shown in Table 6 below.

[0130] Table 6

[0131]

[0132] It should be understood that the disclosed invention is not limited to the specific methods, protocols, and materials described, as these can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is limited only by the appended claims.

[0133] Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. These equivalents are also encompassed by the appended claims.

Claims

1. A method for detecting the genotype of SNP sites in a target gene using cascade amplification for non-diagnostic purposes, characterized in that the method comprises the following steps: Step 1: Design a first primer, a second primer and a mediator probe for the target gene containing the SNP site to be detected, 1.

1. The first primer and the second primer specifically bind to the target gene and are used to amplify the target gene containing the SNP site to be detected; 1.

2. The mediator probe sequentially includes a mediator sequence and a target gene-specific binding sequence from the 5'-end to the 3'-end direction, wherein the mediator sequence cannot bind to the target gene; the target gene-specific binding sequence can specifically and complementarily bind to the target gene, and along the 5'-end to the 3'-end direction, the first base sequence of the target gene-specific binding sequence corresponds to the SNP site of the target gene, and the first base sequence is complementary to the wild-type base or mutant base of the SNP site of the target gene; a group that blocks extension is labeled at the 3'-end of the mediator probe; Step 2: Design an amplification probe according to the mediator sequence of the mediator probe in Step 1. The amplification probe sequentially includes a single-stranded reporter sequence, a double-stranded auxiliary sequence, and a single-stranded mediator sequence-specific binding sequence. The single-stranded reporter sequence does not bind to the target gene, and the single-stranded reporter sequence does not complementarily pair with other sequences in the amplification probe; the double-stranded structure of the auxiliary sequence remains stable during the PCR amplification process; the mediator sequence-specific binding sequence specifically and complementarily binds to the mediator sequence in the mediator probe, and the double-stranded formed after the binding of the mediator sequence and the mediator sequence-specific binding sequence is exactly flush with the double-stranded of the auxiliary sequence, that is, there is no spacer base between the above two double-strands; a group that blocks extension is labeled at the end of the single-stranded mediator sequence-specific binding sequence; Step 3: Design a detection probe according to the single-stranded reporter sequence of the amplification probe. The detection probe sequentially includes a complementary sequence to the single-stranded reporter sequence of the amplification probe, a base complementary to the first base at the 5'-end of the auxiliary sequence, and an extension sequence. For the extension sequence, along the 3'-end to 5'-end direction, the first base cannot be complementary to the second base of the auxiliary sequence along the 5'-end to 3'-end direction; Step 4: In a reaction system containing the first primer, the second primer, the mediator probe, the amplification probe, the detection probe, a nucleic acid sample containing the target gene, and a DNA polymerase, perform PCR amplification and analysis of the amplification product: 4.

1. When the first base of the target gene-specific binding sequence is complementary to the wild-type base of the SNP site of the target gene: 4.1.

1. When the SNP site of the target gene is wild-type, at this time, the first base of the target gene specific binding sequence in the media probe is complementary to the wild-type target gene to form a SNP site base pair. Both the first primer and the second primer are extended and amplified. When extending from the 5'-end to the 3'-end to the last base of the media sequence in the media probe, DNA polymerase will cleave the phosphodiester bond between the SNP site base pair and the first adjacent base pair. The cleaved sequence fragment contains the media sequence and the first base, and this fragment is called the first media primer. This media primer specifically binds to the single-stranded media sequence specific binding sequence of the amplification probe and forms an invasion structure of a single base. After the formation of this structure, DNA polymerase will cut off the single-stranded reporter sequence of the amplification probe together with the invaded first base. The cleaved sequence fragment contains the single-stranded reporter sequence together with the invaded first base, and this sequence fragment is called the first reporter primer; the first reporter primer is completely complementary to the detection probe and extends along the extended sequence part of the detection probe to form a first double-stranded product. When only the formation of the first double-stranded product is detected, it can be determined that the SNP site in the nucleic acid sample containing the target gene is wild-type; 4.1.

2. When the SNP site of the target gene is mutant, at this time, the first base of the target gene specific binding sequence in the media probe is not complementary to the mutant target gene. At this time, neither the first base of the target gene specific binding sequence nor the media sequence is complementary to the target gene. Both the first primer and the second primer are extended and amplified. When extending from the 5'-end to the 3'-end to the first base of the target gene specific binding sequence in the media probe, DNA polymerase will cleave the phosphodiester bond between the first base and the second base that are paired with the target gene in the target gene specific binding sequence. The cleaved sequence fragment contains the media sequence, the first base of the target gene specific binding sequence, and the first base that is paired with the target gene, and this fragment is called the second media primer; The second media primer specifically binds to the amplification probe and forms an invasion structure of two bases. After the formation of this structure, DNA polymerase will cut off the reporter sequence of the amplification probe together with the invaded first and second bases, and this sequence fragment is called the second reporter primer; when the second reporter primer is complementary to the detection probe, the last base at the 3'-end of the second reporter primer cannot be complementary to the detection probe, and the second reporter primer cannot extend along the detection probe. Therefore, a second double-stranded product is obtained. When only the formation of the second double-stranded product is detected, it can be determined that the SNP site in the nucleic acid sample containing the target gene is mutant; 4.1.

3. When the SNP site of the target gene is heterozygous, the first double-stranded product and the second double-stranded product will be generated simultaneously. Therefore, when the formation of both the first double-stranded product and the second double-stranded product is detected, it can be determined that the SNP site in the nucleic acid sample containing the target gene is heterozygous; 4.

2. When the first base sequence of the target gene-specific binding sequence is complementary to the missense mutant base of the SNP site of the target gene: 4.2.

1. If the SNP site of the target gene is wild-type or synonymous mutant, at this time, the first base of the target gene-specific binding sequence in the medium probe does not complementarily bind to the target gene. At this time, neither the first base of the target gene-specific binding sequence nor the medium sequence complementarily binds to the target gene. Just like in 4.1.2 above, only the formation of the second double-stranded product is detected, and it can be determined that the SNP site in the nucleic acid sample containing the target gene is wild-type or synonymous mutant; 4.2.

2. If the SNP site of the target gene is missense mutant, at this time, the first base of the target gene-specific binding sequence in the medium probe complementarily binds to the missense mutant target gene to form an SNP site base pair. Just like in 4.1.1 above, only the formation of the first double-stranded product is detected, and it can be determined that the SNP site in the nucleic acid sample containing the target gene is missense mutant; 4.2.

3. Just like in 4.1.3 above, when the formation of both the first double-stranded product and the second double-stranded product is detected, it can be determined that the SNP site in the nucleic acid sample containing the target gene is heterozygous.

2. The method according to claim 1, characterized in that, When there are multiple SNP sites in the target gene, corresponding medium probes, amplification probes, and detection probes are designed respectively for each SNP site, so that each SNP site forms its respective corresponding first double-stranded product and second double-stranded product.

3. The method according to claim 2, wherein The formation of the first double-stranded product and / or the second double-stranded product is detected by the method of melting curve.

4. The method according to claim 3, characterized in that, The single-stranded reporter sequence of the amplification probe is labeled with a fluorophore; the double-stranded auxiliary sequence of the amplification probe is labeled with a quenching group corresponding to the fluorophore labeled in the single-stranded reporter sequence; the detection probe is modified with a quenching group corresponding to the fluorophore labeled in the single-stranded reporter sequence of the amplification probe, and the formation of the first double-stranded product and / or the second double-stranded product is detected by the method of melting curve.

5. The method according to claim 3, characterized in that The detection probe is labeled with a fluorophore and a corresponding quenching group, and the formation of the first double-stranded product and / or the second double-stranded product is detected by the method of melting curve.

6. The method according to claim 1, characterized in that The sequence of the medium probe, the sequence of the amplification probe, and / or the sequence of the detection probe comprises or consists of naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof.

7. The method according to claim 1, characterized in that, The DNA polymerase includes at least one of Taq DNA polymerase, Tth DNA polymerase, Tfl DNA polymerase, Tfi DNA polymerase, pfu DNA polymerase, KOD DNA polymerase or Tgo DNA polymerase.

8. A kit for detecting the genotype of SNP sites in a target gene by cascade amplification, characterized in that, The kit includes: a DNA polymerase, a first primer, a second primer, a mediator probe, an amplification probe and a detection probe designed for a target gene containing a SNP site to be detected, a. The first primer and the second primer specifically bind to the target gene and are used for amplifying the target gene containing the SNP site to be detected; b. The mediator probe sequentially includes a mediator sequence and a target gene-specific binding sequence from the 5'-end to the 3'-end direction, wherein the mediator sequence cannot bind to the target gene; the target gene-specific binding sequence can specifically and complementarily bind to the target gene, and along the 5'-end to the 3'-end direction, the first base sequence of the target gene-specific binding sequence corresponds to the SNP site of the target gene, and the first base sequence is complementary to the wild-type base or mutant base of the SNP site of the target gene; the 3'-end of the mediator probe is labeled with a group that blocks extension; c. An amplification probe is designed according to the mediator sequence of the mediator probe. The amplification probe sequentially includes a single-stranded reporter sequence, a double-stranded auxiliary sequence and a single-stranded mediator sequence-specific binding sequence. The single-stranded reporter sequence does not bind to the target gene, and the single-stranded reporter sequence does not complementarily pair with other sequences in the amplification probe; the double-stranded structure of the auxiliary sequence remains stable during the PCR amplification process; the mediator sequence-specific binding sequence specifically and complementarily binds to the mediator sequence in the mediator probe, and the double-stranded formed after the binding of the mediator sequence and the mediator sequence-specific binding sequence is exactly flush with the double-stranded of the auxiliary sequence, that is, there is no spacer base between the above two double-strands; the end of the single-stranded mediator sequence-specific binding sequence is labeled with a group that blocks extension; d. A detection probe is designed according to the single-stranded reporter sequence of the amplification probe. The detection probe sequentially includes a complementary sequence to the single-stranded reporter sequence of the amplification probe, a base complementary to the first base at the 5'-end of the auxiliary sequence, and an extension sequence. For the extension sequence, along the 3'-end to 5'-end direction, the first base cannot be complementary to the second base of the auxiliary sequence along the 5'-end to 3'-end direction; the detection probe is modified with a quenching group.

9. The kit according to claim 8, wherein The sequence of the mediator probe, the sequence of the amplification probe and / or the sequence of the detection probe comprises or consists of naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof.

10. The kit according to claim 8, characterized in that, The DNA polymerase includes at least one of Taq DNA polymerase, Tth DNA polymerase, Tfl DNA polymerase, Tfi DNA polymerase, pfu DNA polymerase, KOD DNA polymerase or Tgo DNA polymerase.

11. The kit according to claim 8, wherein The single-stranded reporter sequence of the amplification probe is labeled with a fluorescent group; the double-stranded auxiliary sequence of the amplification probe is labeled with a quenching group corresponding to the fluorescent group labeled in the single-stranded reporter sequence; the detection probe is modified with a quenching group corresponding to the fluorescent group labeled in the single-stranded reporter sequence of the amplification probe, and whether the first double-stranded product and / or the second double-stranded product is formed is detected by the method of melting curve.

12. The kit according to claim 8, wherein The detection probe is labeled with a fluorescent group and a corresponding quenching group, and whether the first double-stranded product and / or the second double-stranded product is formed is detected by the method of melting curve.

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