Method for detecting genotype of SNP site in target gene using cascade amplification, and kit thereof

Abstract

Provided in the present invention are a method for detecting the genotype of an SNP site in a target gene using cascade amplification, and a kit thereof. The method comprises: designing a first primer, a second primer, and a mediator probe for a target gene containing an SNP site to be detected; designing an amplification probe according to a mediator sequence of the mediator probe; designing a detection probe according to a single-stranded reporter sequence of the amplification probe; and performing PCR amplification and analysis of an amplification product in a reaction system comprising 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 target-independent detection method. By means of detecting a melting peak corresponding to the melting point of a certain duplex in a melting curve analysis, the SNP typing of the target gene corresponding to the duplex can be determined.

Description

Method and kit for detecting SNP loci genotypes in target genes using cascade amplification Technical Field

[0001] This invention relates to the field of nucleic acid detection technology, and more specifically, to a method and kit for detecting SNP site genotypes in target genes using cascade amplification. Background Technology

[0002] Drug resistance mutations refer to the genetic mutations that occur in microorganisms (such as bacteria, viruses, and fungi) after exposure to antibiotics or other antimicrobial drugs, leading to reduced or complete loss of drug sensitivity. These mutations may be caused by the drug directly affecting the microorganism's genetic material (such as DNA), or by changes in the microorganism's own repair mechanisms or metabolic pathways. Drug resistance mutations are a common phenomenon in nature and an important mechanism for microorganisms to combat external pressures. However, drug resistance mutations pose a significant challenge to clinical treatment and have become a global public health problem, a major factor seriously threatening human health and safety. Therefore, detecting mutations at drug resistance sites in microbial genes is crucial for guiding clinical drug use, reducing unnecessary drug use, promoting precision medicine, and improving treatment outcomes.

[0003] On the other hand, DNA variations occurring at specific sites on single nucleotides in the genome, known as single nucleotide polymorphisms (SNPs), are among the most common forms of genetic variation in humans. SNPs are closely related to genetic diseases and have a significant impact on regulating gene expression, determining human phenotypes, and regulating metabolic processes. They explain the heritable inter-individual differences in complex phenotypes and the relationship between genes and diseases, and are associated with disease susceptibility, disease pathogenesis, and individual differences in drug response. Therefore, SNP detection can promote the early diagnosis, prevention, and treatment of 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 and real-time quantitative PCR, amplification arrest PCR, molecular beacon method, and high-resolution melting curve method.

[0005] Among them, direct sequencing of PCR products has advantages such as intuitive and reliable results, the ability to analyze unknown DNA sequences and mutation sites, long read range of one-way reaction, and high accuracy. However, the conventional PCR method used in direct sequencing of PCR products has low detection sensitivity, 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 fluorescence signal generated by fluorescence change in one dimension for detection and analysis. For a single SNP site, two specific probes with different fluorescent labels at both ends need to be designed to identify different alleles. For multiplex detection, due to the limitation of the fluorescence channel, it can only be used for the analysis of a small number of SNP sites, making it difficult to distinguish multiple targets. Furthermore, it is relatively difficult to design probes for SNP sites that are close to each other.

[0007] Multicolor melting curve analysis combines multicolor fluorescence with melting curve analysis, performing detection and analysis in two dimensions: fluorescence change and Tm value, achieving multiplex detection in a single channel. This method utilizes the Tm value generated by the hybridization of probes with a large number of DNA single strands for melting curve analysis. Therefore, asymmetric PCR is used. By adjusting the concentration ratio of upstream and downstream primers, a large number of single-stranded products complementary to the fluorescent probe are obtained. The asymmetric amplification is then combined with the DNA melting temperature to form the characteristic peak of the melting curve. This method avoids the limitations of PCR instrument fluorescence channels in other multiplex detection methods, offering advantages such as high throughput, simple operation, low cost, and reliable accuracy. However, multicolor melting curve analysis still faces many challenges. Because asymmetric amplification is linear, non-exponential amplification is prone to low amplification yield and low sensitivity. Furthermore, optimizing the upstream and downstream primer ratio is difficult and design is challenging. The molecular beacon used in asymmetric melting curves undergoes a process of hairpin structure, free single strand, hybridization double strand, and free single strand during the reaction. The fluorescence signal transitions from weak to strong and from strong to weak, which can easily lead to problems such as uneven baseline and inverted peaks.

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

[0009] To address the aforementioned technical problems, this invention provides a method for detecting SNP site genotypes in target genes using cascade amplification, the method comprising the following steps:

[0010] 1. A method for detecting SNP genotypes in target genes using cascade amplification, characterized in that the method comprises the following steps:

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

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

[0013] 1.2. The mediator probe comprises a mediator sequence and a target gene-specific binding sequence sequentially from the 5' end to the 3' end, wherein the mediator sequence cannot bind to the target gene; the target gene-specific binding sequence is specifically complementary to the target gene, and along the 5' end to the 3' end, 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 or mutant base of the SNP site of the target gene; the 3' end of the mediator probe is labeled with a group that prevents elongation.

[0014] Step 2: Design an amplification probe based on the mediator sequence described 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 does not pair complementaryly with other sequences in the amplification probe. The double-stranded structure of the auxiliary sequence remains stable during PCR amplification. The mediator sequence-specific binding sequence binds specifically and complementaryly to the mediator sequence in the mediator probe, and the double strand formed after the mediator sequence binds to the mediator sequence-specific binding sequence is exactly flush with the double strand of the auxiliary sequence, i.e., there is no spacer base between the two double strands. The single-stranded mediator sequence-specific binding sequence is labeled with a group at its end to prevent elongation.

[0015] Step 3: Design a detection probe based on 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. The first base of the extension sequence, along the direction from the 3' end to the 5' end, cannot be complementary to the second base of the auxiliary sequence along the direction from the 5' end to the 3' end.

[0016] Step 4: Perform PCR amplification and analyze the amplification products in a reaction system containing the first primer, the second primer, the medium probe, the amplification probe, the detection probe, the nucleic acid sample containing the target gene, and DNA polymerase.

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

[0018] 4.1.1. If the target gene SNP site is wild-type, then the first base of the target gene-specific binding sequence in the vector probe is complementary to the wild-type target gene to form an SNP site base pair. Both the first primer and the second primer are extended and amplified. When the extension reaches the last base of the vector sequence in the vector probe from the 5' end to the 3' end, DNA polymerase will cleave the phosphodiester bond between the SNP site base pair and its adjacent first base pair. The cleaved sequence fragment contains the vector sequence and the first base. This fragment is called the first vector primer. This vector primer and the amplification probe... The single-stranded mediator sequence specifically binds to the target gene and forms a single-base invasion structure. After this structure is formed, the DNA polymerase cleaves the single-stranded reporter sequence of the amplification probe along with the first invaded base. The cleaved sequence fragment contains the single-stranded reporter sequence along with the first invaded 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 extension sequence 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. If the target gene SNP site is mutant, the first base of the target gene-specific binding sequence in the medium probe does not bind complementaryly to the mutant target gene. In this case, neither the first base of the target gene-specific binding sequence nor the medium sequence binds complementaryly to the target gene. Both the first and second primers are extended amplified. When the extension reaches the first base of the target gene-specific binding sequence in the medium probe from the 5' end to the 3' end, DNA polymerase will cleave the phosphodiester bond between the first and second bases of the target gene-specific binding sequence. The cleaved sequence fragment contains the medium sequence and the first base of the target gene-specific binding sequence. The first base that binds to the target gene and is called the second mediator primer. The second mediator primer specifically binds to the amplification probe and forms an invasion structure of two bases. After the structure is formed, the DNA polymerase cuts off the reporter sequence of the amplification probe along with the invaded first and second bases. This sequence fragment is called the second reporter primer. When the second reporter primer binds complementary to the detection probe, the last base at the 3' end of the second reporter primer cannot bind 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 a mutant.

[0020] 4.1.3. If the target gene SNP site is heterozygous, the first double-stranded product and the second double-stranded product will be generated simultaneously. Therefore, when the first double-stranded product and the second double-stranded product are detected to be formed simultaneously, 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 a missense mutant base at the SNP site of the target gene:

[0022] 4.2.1. If the target gene SNP site is wild-type or synonymous mutant, then the first base of the target gene-specific binding sequence in the medium probe does not bind complementaryly to the target gene. In this case, neither the first base of the target gene-specific binding sequence nor the medium sequence binds complementaryly to the target gene. As in 4.1.2 above, only the formation of the second double-stranded product is detected, which can determine 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 target gene SNP site is a missense mutant, then the first base of the target gene-specific binding sequence in the medium probe is complementary to the missense mutant target gene to form an SNP site base pair. As in 4.1.1 above, as long as 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. As in 4.1.3 above, when the first double-stranded product and the second double-stranded product are detected to be formed simultaneously, it can be determined that the SNP site in the nucleic acid sample containing the target gene is heterozygous.

[0025] In one embodiment, when the target gene contains multiple SNP sites, a corresponding mediator probe, amplification probe, and detection probe are designed for each SNP site, so that each SNP site forms its own first double-stranded product and second double-stranded product.

[0026] In one embodiment, the formation of the first double-chain product and / or the second double-chain product is detected by a melting curve method.

[0027] In one embodiment, 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 the formation of the first double-stranded product and / or the second double-stranded product is detected by melting curve method.

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

[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. The kit includes: a DNA polymerase, a first primer designed for a target gene containing the SNP site to be tested, a second primer and a medium probe, an amplification probe and a detection probe.

[0032] a. The first primer and the second primer specifically bind to the target gene to amplify the target gene containing the SNP site to be tested;

[0033] b. The mediator probe comprises, from the 5' end to the 3' end, a mediator sequence and a target gene-specific binding sequence, wherein the mediator sequence cannot bind to the target gene; the target gene-specific binding sequence is specifically complementary to the target gene, and along the 5' end to the 3' end, 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 or mutant base of the SNP site of the target gene; the 3' end of the mediator probe is labeled with a group that prevents elongation.

[0034] c. An amplification probe is designed based on 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 it does not pair complementaryly with other sequences in the amplification probe. The double-stranded structure of the auxiliary sequence remains stable during PCR amplification. The mediator sequence-specific binding sequence binds specifically and complementaryly to the mediator sequence in the mediator probe, and the double strand formed after the mediator sequence binds to the mediator sequence-specific binding sequence is exactly flush with the double strand of the auxiliary sequence, i.e., there is no spacer base between the two double strands. The end of the single-stranded mediator sequence-specific binding sequence is labeled with a group that prevents elongation.

[0035] d. Design a detection probe based on 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. The first base of the extension sequence, along the direction from the 3' end to the 5' end, cannot be complementary to the second base of the auxiliary sequence along the direction from the 5' end to the 3' end.

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

[0037] 1. The method of this invention comprises two amplification cycles. The first amplification cycle is PCR amplification, in which mediator primers are continuously generated as the PCR amplification reaction proceeds. The second amplification cycle involves the binding of the mediator primers to the amplification probe, cleaving and releasing the reporter primer. Subsequently, the reporter primer continuously binds to the detection probe, forming a double-stranded product. In the second amplification cycle, the mediator primers are not consumed but act as a catalyst, continuously binding to the amplification probe and cleaving and releasing the reporter primer as the amplification reaction proceeds. However, the two amplification cycles are interconnected yet 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 achieving cascade amplification of the target gene and further improving detection sensitivity.

[0038] 2. The method of this invention is a target-independent detection method, meaning that the sequences of each reporter primer and detection probe are artificially designed, known, or predetermined. Therefore, the first double-stranded product and the second double-stranded product are determinable. In melting curve detection, the melting point (Tm value) of each double-stranded compound can be pre-calculated. Thus, by detecting the melting peak with the melting point (Tm value) of a certain double-stranded compound in melting curve analysis, the SNP genotype of the target gene corresponding to that double-stranded compound can be determined. This solves the problem of melting curve peak shift and misjudgment caused by the easy mutation of the target gene sequence, and effectively addresses the issue that melting curve analysis methods are not suitable for RNA samples.

[0039] 3. Currently, the widely used asymmetric melting curve method designs mutation sites onto probes and uses the difference in Tm values ​​of a single fluorescent probe to distinguish between wild-type and mutant types. For ATGC>GC mutations, the difference in Tm values ​​is easily distinguishable, but for A>T mutations, the difference in Tm values ​​at probe binding is smaller, making genotype differentiation difficult. Asymmetric melting curves are currently widely used in SNP genotyping. This method designs mutation sites onto probes. Because the probes are non-selective, only one probe is designed for each SNP site to distinguish between two genotypes. Genotype differentiation is achieved by using the single-base Tm value at which the probe binds to the single strand in the same fluorescent channel. For ATGC>GC mutations, the difference in Tm values ​​at probe binding is easily distinguishable, but for A>T mutations, the difference in Tm values ​​at probe binding is smaller, making genotype differentiation difficult. Furthermore, due to limitations in probe temperature and the two peaks per channel, a maximum of 2-3 SNP sites can be designed for one fluorescent channel. The method described in this invention designs the first base of the target gene-specific binding sequence of the mediator probe as an SNP site. By utilizing whether the target gene SNP site binds complementary to the mediator probe, two different mediator primers are generated from the cleavage. Due to differences in the 3' terminal sequences, these different mediator primers form invasion structures with different base numbers after binding to the amplification probe. These different invasion structures generate two different reporter primers. Due to differences in the 3' terminal sequences, after binding to the detection probe, only one reporter primer can extend along the detection probe, while the other reporter primer can only bind to the detection probe but cannot extend. Therefore, the melting point (Tm value) of the double strand can be arbitrarily constructed, enabling customization of the SNP site melting curve peak. This solves the problem of indistinguishable Tm values ​​between wild-type and mutant genotypes of the same SNP site, reduces design difficulty, and allows for the specific differentiation of A>T base mutations at the target site. Different SNP sites are distinguished by controlling the amplicon length and GC content between the reporter primer and the detection probe, and different SNP sites are distinguished by the Tm value of different melting curves, thus increasing the number of detectable SNP sites.

[0040] 4. In the method of the present invention, the design of the double-stranded auxiliary sequence of the amplification probe cleverly forms a large spatial steric hindrance, which prevents excessive amplification probe from combining with the detection probe to generate false positive background peaks, thus avoiding the problem of false positive background peaks interfering with the accuracy of interpretation.

[0041] 5. In the method of this invention, when using the melting curve method to detect the first double-stranded product and the second-stranded product, the fluorescence signal fluctuations of the reporter primer and the detection probe are reflected to a greater extent during the melting curve analysis, resulting in a flatter baseline, reducing the occurrence of non-specific peaks, and improving the accuracy and precision of the detection method. Simultaneously, this method is not limited by the number of fluorescence channels that can be detected; it distinguishes different target genes based on melting point and fluorescence color, increasing the number of targets detected per well and improving detection throughput. The method of this invention allows for multiple target detection, with the melting peak baseline remaining flat, higher peak values, and no non-specific peaks, resulting in higher sensitivity; at the same time, the detection range is also wider, and the melting curve detection T... m The value range is 40~80℃;

[0042] 6. When using the melting curve method for detection, the method for detecting target nucleic acids in this invention is a symmetrical amplification melting curve method. Compared with the common asymmetric amplification melting curve method (a linear amplification method), this method utilizes the advantages of PCR exponential amplification to enrich a large amount of target nucleic acids and improve detection sensitivity. Attached Figure Description

[0043] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments recorded in this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0044] Figure 1 is a schematic diagram of the magnified probe used in the method of the present invention;

[0045] Figure 2 is a schematic diagram illustrating the basic principle of the method of the present invention;

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

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

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

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

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

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

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

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

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

[0055] Figure 12 shows the melting curve analysis of MTHFR(677C>T) as the wild type and MTHFR(1298A>C) as the heterozygous type;

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

[0057] Figure 14 shows the melting curve analysis of MTHFR(677C>T) and MTHFR(1298A>C), which are both heterozygous. Detailed Implementation

[0058] 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 merely some embodiments of this application, and not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this application. Unless otherwise specified, the following embodiments are all conventional methods in the art.

[0059] Example 1: Basic Principles of the Invention

[0060] As shown in Figures 1 and 2, the present invention provides a method for detecting the genotype of SNP sites in a target gene using cascade amplification, the method comprising the following steps:

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

[0062] 1.1. The first primer and the second primer specifically bind to the target gene to amplify the target gene containing the SNP site to be tested;

[0063] 1.2. The mediator probe comprises a mediator sequence and a target gene-specific binding sequence sequentially from the 5' end to the 3' end, wherein the mediator sequence cannot bind to the target gene; the target gene-specific binding sequence is specifically complementary to the target gene, and along the 5' end to the 3' end, 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 or mutant base of the SNP site of the target gene; the 3' end of the mediator probe is labeled with a group that prevents elongation.

[0064] Step 2: Design an amplification probe based on the mediator sequence described in Step 1, as shown in Figure 1a. The amplification probe of this invention can be designed in various forms. The amplification probe can consist of one strand, 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 pair complementaryly with other sequences in the amplification probe. The double-stranded structure of the auxiliary sequence remains stable during PCR amplification. The mediator sequence-specific binding sequence specifically binds complementaryly to the mediator sequence in the mediator probe, and the double strand formed after the mediator sequence binds to the mediator sequence-specific binding sequence is exactly flush with the double strand of the auxiliary sequence, i.e., there is no spacer base between the two double strands (as shown in Figure 1b). The end of the single-stranded mediator sequence-specific binding sequence is labeled with a group that prevents elongation.

[0065] Step 3: Design a detection probe based on 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. The first base of the extension sequence, along the direction from the 3' end to the 5' end, cannot be complementary to the second base of the auxiliary sequence along the direction from the 5' end to the 3' end.

[0066] Step 4: Perform PCR amplification and analyze the amplification products in a reaction system containing the upstream first primer, second primer, mediator probe, amplification probe, detection probe, nucleic acid sample containing the target gene, and DNA polymerase.

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

[0068] 4.1.1. If the target gene SNP site is wild-type, then the first base of the target gene-specific binding sequence in the vector probe is complementary to the wild-type target gene to form an SNP site base pair. Both the first primer and the second primer are extended and amplified. When the extension reaches the last base of the vector sequence in the vector probe from the 5' end to the 3' end, DNA polymerase will cleave the phosphodiester bond between the SNP site base pair and its adjacent first base pair. The cleaved sequence fragment contains the vector sequence and the first base. This fragment is called the first vector primer. This vector primer is used in conjunction with the single strand of the amplification probe. The mediator specifically binds to the target sequence and forms a single-base invasion structure (as shown in Figure 1c). After this structure is formed, the DNA polymerase cleaves the single-stranded reporter sequence of the amplification probe along with the first invaded base. The cleaved sequence fragment contains the single-stranded reporter sequence along with the first invaded 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 extension sequence 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. If the target gene SNP site is mutant, then the first base of the target gene-specific binding sequence in the medium probe does not bind complementaryly to the mutant target gene. In this case, neither the first base of the target gene-specific binding sequence nor the medium sequence binds complementaryly to the target gene. Both the first and second primers are extended amplified. When the extension reaches the first base of the target gene-specific binding sequence in the medium probe from the 5' end to the 3' end, DNA polymerase will cleave the phosphodiester bond between the first and second bases of the target gene-specific binding sequence. The cleaved sequence fragment contains the medium sequence and the first base of the target gene-specific binding sequence. The first base that pairs with and binds to the target gene is called the second mediator primer. The second mediator primer specifically binds to the amplification probe and forms an invasion structure of two bases. After the structure is formed, the DNA polymerase cleaves the reporter sequence of the amplification probe along with the invaded first and second bases. This sequence fragment is called the second reporter primer. When the second reporter primer binds complementary to the detection probe, the last base at the 3' end of the second reporter primer cannot bind 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 a mutant.

[0070] 4.1.3. If the target gene SNP site is heterozygous, the first double-stranded product and the second double-stranded product will be generated simultaneously. Therefore, when the first double-stranded product and the second double-stranded product are detected to be formed 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 a missense mutant base at the SNP site of the target gene:

[0072] 4.2.1. If the target gene SNP site is wild-type or synonymous mutant, then the first base of the target gene-specific binding sequence in the medium probe does not bind complementaryly to the target gene. In this case, neither the first base of the target gene-specific binding sequence nor the medium sequence binds complementaryly to the target gene. As in 4.1.2 above, only the formation of the second double-stranded product is detected, which can determine that the SNP site in the nucleic acid sample containing the target gene is wild-type or synonymous mutant.

[0073] 4.2.2. If the target gene SNP site is a missense mutant, then the first base of the target gene-specific binding sequence in the medium probe is complementary to the missense mutant target gene to form an SNP site base pair. As in 4.1.1 above, as long as 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.

[0074] 4.2.3. As in 4.1.3 above, when the first double-stranded product and the second double-stranded product are detected to be formed simultaneously, it can be determined that the SNP site in the nucleic acid sample containing the target gene is heterozygous.

[0075] In the method of the present invention, 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 the formation of the first double-stranded product and / or the second double-stranded product is detected by melting curve method.

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

[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 may bind complementaryly to either the sense or antisense strand of the double-stranded auxiliary sequence of the amplification probe, or it may not bind complementaryly to any strand of the double-stranded auxiliary sequence of the amplification probe.

[0079] In this invention, because the base sequence and length of the detection probe can be pre-designed, the first double-stranded product obtained by extending 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 Similarly, the second double-stranded product obtained by binding the second reporter primer to the detection probe also has a predictable length and structure, and 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, therefore T m1 >T m2 The double-stranded product obtained in step four is subjected to melting curve analysis. By detecting the peak position of the melting curve of the double-stranded product, the corresponding genotype can be determined.

[0080] In this invention, the fluorescent group includes, but is not limited to, various commonly used 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, but is not limited to, various commonly used quenching agents such as Dabcyl, Eclipse, BHQ-1, BHQ-2, QYS-7, etc.

[0081] Example 2: Genotyping of the MTHFR C677T locus

[0082] This embodiment takes the detection of the MTHFR C677T site as an example, and uses the method of the present invention to detect the MTHFR gene. The specific method includes the following steps.

[0083] I. Primer and probe sequence information

[0084] The first primer, second primer, mediator probe, amplification probe, and detection probe are designed based on the target nucleic acid site to be tested. Among them, the mediator probe is designed based on the first base sequence of the target gene-specific binding sequence being complementary to the missense mutant base of the target gene SNP site. The sequence information is shown in Table 1 below.

[0085] Table 1 Note: The bolded part of the probe is the mediator sequence, the italicized part is the SNP site, and the lowercase letters are the target gene-specific sequences; the bolded part of the magnified probe is the reporter sequence, the uppercase letters underlined parts are the double-stranded auxiliary sequences, and the italicized parts are the mediator sequence-specific binding sequences.

[0086] II. PCR Amplification Reaction System and Procedure

[0087] This 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... HS Multiplex Taq DNA Polymerase, 0.1 mM dNTPs, 40 nM first primer, 40 nM second primer, 20 nM medium probe, 100 nM amplification probe, 100 nM detection probe, 5 μL nucleic acid template.

[0088] The fluorescence PCR reaction program of this invention is as follows: pre-denaturation at 95℃ for 5 min; denaturation at 95℃ for 15 s; annealing and extension at 60℃ for 45 s, with fluorescence collected simultaneously, repeated for 45 cycles; the melting curve program is as follows: 95℃ for 2 min, 40℃ for 5 min, and melting curve analysis at 40-80℃, with fluorescence signal detected every 0.04℃. The instrument used in this embodiment is a SLAN 96 real-time fluorescence PCR instrument (Shanghai Hongshi Medical Technology Co., Ltd.).

[0089] III. Melting Curve Analysis

[0090] The results of the melting curve analysis are shown in Table 2 below.

[0091] Table 2

[0092] Targeting the MTHFR C677T mutation site, the VIC channel in T m A peak was observed at 54.5±1℃, indicating a wild-type peak, as shown in Figure 3; the VIC channel showed a peak at T... m A peak was observed at 67.1±1℃, indicating a homozygous mutant, as shown in Figure 4; the VIC channel showed a peak at T... m The peaks at 54.6±1℃ and 67.3±1℃ are observed simultaneously, indicating a heterozygous type, as shown in Figure 5.

[0093] Example 3: Genotyping of the MTHFR A1298C locus

[0094] This embodiment takes the detection of the MTHFR A1298C mutation site as an example, and uses the method of the present invention to detect mutations in the MTHFR gene. The specific method includes the following steps.

[0095] I. Primer and probe sequence information

[0096] The first primer, second primer, mediator probe, amplification probe, and detection probe are designed based on the target nucleic acid site to be tested. Among them, the mediator probe is designed based on the first base sequence of the target gene-specific binding sequence being complementary to the missense mutant base of the target gene SNP site. The sequence information is shown in Table 3 below.

[0097] Table 3

[0098] Note: The bolded part of the probe is the mediator sequence, the italicized part is the SNP site, and the lowercase letters are the target gene-specific sequences; the bolded part of the magnified probe is the reporter sequence, the uppercase letters with underlined parts are the double-stranded auxiliary sequences, and the italicized parts are the mediator sequence-specific binding sequences.

[0099] II. PCR Amplification Reaction System and Procedure

[0100] This 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... HS Multiplex Taq DNA Polymerase, 0.1 mM dNTPs, 40 nM first primer, 40 nM second primer, 20 nM medium probe, 100 nM amplification probe, 100 nM detection probe, 2, 5 μL nucleic acid template.

[0101] The fluorescence PCR reaction program of this invention is as follows: pre-denaturation at 95℃ for 5 min; denaturation at 95℃ for 15 s; annealing and extension at 60℃ for 45 s, with fluorescence collected simultaneously, repeated for 45 cycles; the melting curve program is as follows: 95℃ for 2 min, 40℃ for 5 min, and melting curve analysis at 40-80℃, with fluorescence signal detected every 0.04℃. The instrument used in this embodiment is a SLAN 96 real-time fluorescence PCR instrument (Shanghai Hongshi Medical Technology Co., Ltd.).

[0102] III. Melting Curve Analysis

[0103] The results of the melting curve analysis are shown in Table 4 below.

[0104] Table 4

[0105] Targeting the MTHFR A1298C mutation site, the ROX channel in T m A peak appears at 48.0±1℃, indicating a wild-type peak, as shown in Figure 6; the ROX channel at T... m A peak was observed at 65.3±1℃, indicating a homozygous mutant, as shown in Figure 7; the ROX channel showed a peak at T... m The peaks at 48.1±1℃ and 65.2±1℃ are observed simultaneously, indicating a heterozygous type, as shown in Figure 8.

[0106] Example 3: MTHFR C677T and A1298C two-site genotyping

[0107] This embodiment takes the detection of the MTHFR C677T and MTHFR A1298C double mutation sites as an example, and uses the method of the present invention to detect mutations in the MTHFR gene. The specific method includes the following steps:

[0108] I. Primer and probe sequence information

[0109] First primer, second primer, mediator probe, amplification probe and detection probe were designed according to the target nucleic acid site to be tested. Among them, the mediator probe was designed based on the first base sequence of the target gene-specific binding sequence being complementary to the missense mutant base of the target gene SNP site. The sequence information is shown in Table 5 below.

[0110] Table 5 Note: The bolded part of the probe is the mediator sequence, the italicized part is the SNP site, and the lowercase letters are the target gene-specific sequences; the bolded part of the magnified probe is the reporter sequence, the uppercase letters with underlined parts are the double-stranded auxiliary sequences, and the italicized parts are the mediator sequence-specific binding sequences.

[0111] II. PCR Amplification Reaction System and Procedure

[0112] This 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... HS Multiplex Taq DNA Polymerase, 0.2 mM dNTPs, 40 nM first primer, 40 nM second primer, 20 nM medium probe, 100 nM amplification probe, 100 nM detection probe 1 and 100 nM detection probe 2, 5 μL nucleic acid template.

[0113] The fluorescence PCR reaction program of this invention is as follows: pre-denaturation at 95℃ for 5 min; denaturation at 95℃ for 15 s; annealing and extension at 60℃ for 45 s, with fluorescence collected simultaneously, repeated for 45 cycles; the melting curve program is as follows: 95℃ for 2 min, 40℃ for 5 min, and melting curve analysis at 40-80℃, with fluorescence signal detected every 0.04℃. The instrument used in this embodiment is a SLAN 96 real-time fluorescence PCR instrument (Shanghai Hongshi Medical Technology Co., Ltd.).

[0114] For the dual-site single-tube detection of MTHFR C677T and MTHFR A1298C, the results of the random template melting curves are shown in Figures 9 to 14.

[0115] a. If the VIC channel only shows a peak at 67.3±1℃ and the ROX channel only shows a peak at 65.2±1℃, then both sites are homozygous mutants (Figure 9).

[0116] b. If the VIC channel only shows a peak at 54.6±1℃ and the ROX channel only shows a peak at 48.1±1℃, then both sites are wild-type (Figure 10).

[0117] c. If the VIC channel only shows a peak at 54.6±1℃ and the ROX channel only shows a peak at 65.2±1℃, then C677T is wild-type and the A1298C site is homozygous mutant (Figure 11).

[0118] d. If the VIC channel only peaks at 54.6±1℃, and the ROX channel peaks at both 48.1±1℃ and 65.2±1℃, then C677T is wild-type and the A1298C site is a heterozygous mutant (Figure 12).

[0119] e. If the VIC channel shows peaks at both 54.6±1℃ and 67.3±1℃, and the ROX channel shows a peak only at 65.2±1℃, then C677T is a heterozygous mutant and A1298C is a homozygous mutant (Figure 13).

[0120] f. If the VIC channel shows peaks at 54.6±1℃ and 67.3±1℃ simultaneously, and the ROX channel shows peaks at 48.1±1℃ and 65.2±1℃ simultaneously, then the C677T and A1298C sites are both heterozygous mutants (Figure 14).

[0121] III. Melting Curve Analysis

[0122] The results of the melting curve analysis are shown in Table 6 below.

[0123] Table 6

[0124] It should be understood that the disclosed invention is not limited to the specific methods, schemes, and substances described, as these are all subject to variation. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the invention, which is limited only by the appended claims.

[0125] Those skilled in the art will also recognize, or be able to identify, many equivalents of the specific embodiments of the invention described herein using no more than conventional experiments. These equivalents are also included in the appended claims.

Claims

1. A method for detecting SNP genotypes in target genes using cascade amplification, characterized in that the method comprises the following steps: Step 1: Design the first primer, second primer, and vector probe for the target gene containing the SNP site to be tested. 1.

1. the first primer and the second primer specifically bind to the target gene for amplifying the target gene containing the SNP site to be detected; 1.

2. the mediator probe comprises, in order from 5' end to 3' end, a mediator sequence and a target gene specific binding sequence, wherein the mediator sequence cannot bind to the target gene; the target gene specific binding sequence can specifically bind to the target gene in complement, and the first base sequence of the target gene specific binding sequence corresponds to the SNP site of the target gene along the direction from 5' end to 3' end, 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 an extension-preventing group; Step 2: designing an amplification probe according to the mediator sequence of the mediator probe in step 1, the amplification probe comprises, in order, 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, 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 PCR amplification; the mediator sequence specific binding sequence specifically complements the mediator sequence in the mediator probe, and the double-stranded structure formed after the mediator sequence specifically binds to the mediator sequence specific binding sequence is exactly flush with the double-stranded structure of the auxiliary sequence, i.e. there is no spacer base between the two double-stranded structures; the end of the single-stranded mediator sequence specific binding sequence is labeled with an extension-preventing group; Step 3: designing a detection probe according to the single-stranded reporter sequence of the amplification probe, the detection probe comprises, in order, a complementary sequence of 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, the first base along the direction from 3' end to 5' end cannot complement the second base of the auxiliary sequence along the direction from 5' end to 3' end; Step 4: performing PCR amplification and analysis of the amplification product 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: 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.

2. when the first base of the target gene specific binding sequence is complementary to the mutant base of the SNP site of the target gene: 4.1.

1. If the target gene SNP site is wild-type, then the first base of the target gene-specific binding sequence in the vector probe is complementary to the wild-type target gene to form an SNP site base pair. Both the first primer and the second primer are extended and amplified. When the extension reaches the last base of the vector sequence in the vector probe from the 5' end to the 3' end, DNA polymerase will cleave the phosphodiester bond between the SNP site base pair and its adjacent first base pair. The cleaved sequence fragment contains the vector sequence and the first base. This fragment is called the first vector primer. This vector primer and the amplification probe... The single-stranded mediator sequence specifically binds to the target gene and forms a single-base invasion structure. After this structure is formed, the DNA polymerase cleaves the single-stranded reporter sequence of the amplification probe along with the first invaded base. The cleaved sequence fragment contains the single-stranded reporter sequence along with the first invaded 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 extension sequence 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. If the target gene SNP site is mutant, the first base of the target gene-specific binding sequence in the medium probe does not bind complementaryly to the mutant target gene. In this case, neither the first base of the target gene-specific binding sequence nor the medium sequence binds complementaryly to the target gene. Both the first primer and the second primer are extended and amplified. When the extension reaches the first base of the target gene-specific binding sequence in the medium probe from the 5' end to the 3' end, the DNA polymerase will cut the phosphodiester bond between the first base and the second base of the target gene-specific binding sequence. The cut sequence fragment contains the medium sequence, the first base of the target gene-specific binding sequence, and the first base of the target gene. This fragment is called the second medium primer. The second mediator primer specifically binds to the amplification probe and forms a two-base invasion structure. After this structure is formed, the DNA polymerase cleaves the reporter sequence of the amplification probe along with the first and second invaded bases. This sequence fragment is called the second reporter primer. When the second reporter primer binds complementary to the detection probe, the last base at the 3' end of the second reporter primer cannot bind 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 a mutant. 4.1.

3. If the target gene SNP site is heterozygous, the first double-stranded product and the second double-stranded product will be generated simultaneously. Therefore, when the first double-stranded product and the second double-stranded product are detected to be formed simultaneously, 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 a missense mutant base at the SNP site of the target gene: 4.2.

1. If the target gene SNP site is wild-type or synonymous mutant, then the first base of the target gene-specific binding sequence in the medium probe does not bind complementaryly to the target gene. In this case, neither the first base of the target gene-specific binding sequence nor the medium sequence binds complementaryly to the target gene. As in 4.1.2 above, only the formation of the second double-stranded product is detected, which can determine that the SNP site in the nucleic acid sample containing the target gene is wild-type or synonymous mutant. 4.2.

2. If the target gene SNP site is a missense mutant, then the first base of the target gene-specific binding sequence in the medium probe is complementary to the missense mutant target gene to form an SNP site base pair. As in 4.1.1 above, as long as 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. 4.2.

3. As in 4.1.3 above, when the first double-stranded product and the second double-stranded product are detected to be formed simultaneously, it can be determined that the SNP site in the nucleic acid sample containing the target gene is heterozygous.

2. The method of claim 1, wherein, When the target gene contains multiple SNP sites, corresponding mediator probes, amplification probes and detection probes are designed for each SNP site, so that each SNP site forms its own first double-stranded product and second double-stranded product.

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

4. The method of claim 3, wherein, The amplification probe has a fluorescent group labeled in its single-stranded reporter sequence; the amplification probe has a quenching group labeled in its double-stranded auxiliary sequence 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 the formation of the first double-stranded product and / or the second double-stranded product is detected by melting curve method.

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

6. The method of claim 1, wherein, The mediator probe sequence, the amplification probe, and / or the detection probe sequence comprise or consist of naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof.

7. The method of claim 1, wherein, 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 a SNP site in a target gene using cascade amplification, characterized in that, The kit includes: DNA polymerase, a first primer, a second primer and a medium probe designed for a target gene containing the SNP site to be tested, an amplification probe and a detection probe. a. The first primer and the second primer specifically bind to the target gene to amplify the target gene containing the SNP site to be tested; b. The mediator probe comprises, from the 5' end to the 3' end, a mediator sequence and a target gene-specific binding sequence, wherein the mediator sequence cannot bind to the target gene; the target gene-specific binding sequence is specifically complementary to the target gene, and along the 5' end to the 3' end, 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 or mutant base of the SNP site of the target gene; the 3' end of the mediator probe is labeled with a group that prevents elongation. c. An amplification probe is designed based on 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 it does not pair complementaryly with other sequences in the amplification probe. The double-stranded structure of the auxiliary sequence remains stable during PCR amplification. The mediator sequence-specific binding sequence binds specifically and complementaryly to the mediator sequence in the mediator probe, and the double strand formed after the mediator sequence binds to the mediator sequence-specific binding sequence is exactly flush with the double strand of the auxiliary sequence, i.e., there is no spacer base between the two double strands. The end of the single-stranded mediator sequence-specific binding sequence is labeled with a group that prevents elongation. d. Design a detection probe based on 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. The first base of the extension sequence, along the direction from the 3' end to the 5' end, cannot be complementary to the second base of the auxiliary sequence along the direction from the 5' end to the 3' end. The detection probe is modified with a quenching group.

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

10. The kit of claim 8, wherein 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 of 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 quencher group corresponding to the fluorescent group labeled in the single-stranded reporter sequence; the detection probe is modified with a quencher 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 a melting curve method.

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

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