CN114457165A - Primer probe set, kit and detection method for detecting HLA-A02 gene - Google Patents

Abstract

The invention discloses a primer probe set, a kit and a detection method for detecting HLA-A02 gene; wherein the primer probe group comprises HLA-A02 specific primers and HLA-A02 specific probes, the nucleotides of which are shown as SEQ ID NO.1 to SEQ ID NO. 6; the detection method of the HLA-A02 gene is realized by adopting a kit to detect HLA-A02 based on TaqMan probe PCR technology, and has very high specificity; meanwhile, the method effectively avoids the interaction between primers, between primers and probes and between probes through reasonable primer and probe matching, improves the specificity of HLA-A02 detection, and effectively avoids false positive.

Description

Primer probe set, kit and detection method for detecting HLA-A02 gene

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a primer probe set, a kit and a detection method for detecting HLA-A02 gene.

Background

Cancer immunotherapy utilizes the immune system to combat cancer. Although immunotherapy has been an important component of cancer therapy for decades, it has not been of interest until the last decade, particularly after the nobel physiological and medical prizes were awarded for the discovery of cytotoxic T-lymphocyte-associated protein (CTLA-4) and programmed cell death protein-1/programmed cell death protein ligand-1 (PD-1/PD-L1). Immune checkpoint inhibitors are now being used as first line therapy for the treatment of a variety of cancers, including advanced non-small cell lung cancer (NSCLC), melanoma, and renal cell carcinoma. In addition to immune checkpoint inhibitors, adoptive cell therapy and tumor vaccines are also common cancer immunotherapies. The basic mechanism of these cancer immunotherapies is that T cells exert immune functions by recognizing tumor antigens presented by Major Histocompatibility Complexes (MHC) on the tumor cell membrane. MHC is a group of polymorphic genes expressed in almost all vertebrates that determines histocompatibility between different individuals. MHC was first discovered in the first decade of the 20 th century due to tumor rejection between genetically diverse mice. Human MHC is also known as Human Leukocyte Antigen (HLA). HLA class i molecules are expressed on most cell types, including human tumor cells, presenting endogenous antigens to the immune system. HLA class ii molecules are expressed primarily by Antigen Presenting Cells (APCs), presenting exogenous antigens to T helper cells. Both HLA class I and II molecules show a high degree of polymorphism, which means that they have many different alleles in the human population. This is why HLA mismatch between donor and recipient is the main reason for transplant rejection. Based on the IMGT/HLA database, over 12,000 alleles were identified as HLA class I genes. Evolutionarily, the diversity of HLA molecules ensures that the immune system is able to recognize as many antigens as possible and helps us defend against various pathogens. Interestingly, in studying the effect of HLA class I differences on the efficacy of immune checkpoint inhibitors in the treatment of cancer, Chofell et al found that greater sequence differences in HLA class I genotypes were associated with greater diversity in self, tumor and viral immunopeptide groups. In addition, patients with high HLA class i differences respond better to immune checkpoint inhibitors than patients with low HLA class i differences. These findings indicate that HLA polymorphisms are critical for our fighting against cancer.

CD8⁺T cells (often referred to as cytotoxic T lymphocytes or CTLs) are an important component of the adaptive immune system against tumors. Most cytotoxic T cells express a T Cell Receptor (TCR) that can recognize a specific antigen. After the TCR binds to the peptide-HLA complex expressed by the tumor cells, CTLs are activated, accumulate at the tumor site, releasing effectors to attack the tumor cells. CTLs exert specific killing effects on tumor cells through two major pathways: (ii) (one) release of cytotoxic substances such as perforin, granzyme and cytokines to kill tumor cells; (II) activating death receptor pathway, such as FasL expressed on T cell surface, and combining with death receptor Fas on tumor cell surface, initiating apoptosis-related signal, leading to tumor cell apoptosis. Recently, with the aid of genetic engineering techniques such as lentiviruses, specific TCRs recognizing tumor antigens have been inserted into the genomes of a large number of T cells, which has made it possible to produce antigen-specific T cells for everyone. TCR-engineered T cells (TCR-T) are therefore considered to be a more promising approach to cancer patient treatment. The peptide-HLA complex recognized by the TCR is called a T cell epitope. A very limited number of T-cell epitopes have been identified in clinical trials and used in immunotherapy, and most of the known peptides are restricted by antigens such as HLA-A02 and HLA-A02, so that the detection of HLA-A02 in patients is of great significance in improving the therapeutic effect of cancer.

The current typing methods for HLA are mainly of type 3: serological, cytological and molecular biology typing methods. Among them, serological and cytological typing methods are rarely used at present because of the difficulty in obtaining highly specific antibodies and specific cells and the cumbersome operation. With the development of molecular biology techniques, rapid and accurate HLA genotyping established at the DNA molecular level has gradually replaced traditional serotyping methods. The molecular biology typing method mainly comprises polymerase chain reaction-allele genome specific primers (PCR-SSP), polymerase chain reaction-sequence specific oligonucleotide probe (PCR-SSO), polymerase reaction-direct sequencing method (PCR-SBT) and fluorescent quantitative PCR (qPCR). The PCR-SBT can directly determine the sequence of the HLA genotype, has high resolution and can directly discover new alleles, but because of heterozygotes, accurate results of each haplotype can be obtained only after homologous chromosomes are separated, the complexity of operation is increased, and the detection cost is correspondingly higher; PCR-SSP and PCR-SSO belong to low-resolution HLA typing methods, wherein the PCR-SSP method is analyzed by amplifying DNA through a plurality of pairs of specific primers and an electrophoresis result, has large pollution risk and long period, and is not suitable for clinical application at present; the current fluorescence PCR detection method has the main advantages of (1) closed tube operation and closed state detection, and reduces the possibility of template pollution and false positive; (2) post-treatment of PCR products is not needed, so that the operation is simpler, more convenient and faster; (3) the sensitivity is high, and single copy genes can be detected; (4) the specificity is high, and the fluorescence PCR can specifically detect the target gene by the specific combination of the Taqman probe and the target sequence.

The current method for detecting HLA-A02 cannot meet the requirements of high efficiency, accuracy, simplicity and convenience and the like, and the detection time of a large number of samples is long and tedious, so that a method which is rapid, effective, accurate and suitable for detecting HLA-A02 by using a large number of samples is urgently needed.

Disclosure of Invention

Aiming at the defects in the prior art, the invention designs a primer probe set, a kit and a detection method for detecting HLA-A02 based on the latest scientific research dynamics and clinical application. The detection method is simple, convenient, rapid, accurate and effective, has simple and easily-distinguished result, and is suitable for detecting mass samples.

The technical scheme of the invention is as follows:

a primer probe group for detecting HLA-A02 gene comprises a primer probe group with nucleotides shown as SEQ ID No.1 to SEQ ID No.6, and the primer probe group comprises an HLA-A02 specific primer and a probe; wherein the primer probe sets shown in SEQ ID NO.1 to SEQ ID NO.6 comprise the following components:

an upstream primer SEQ ID NO.1: 5'-TCCTCGTCCCCAGGCACT-3';

a downstream primer of SEQ ID NO.2: 5'-GAACTGCGTGTCGTCCACG-3';

an upstream primer SEQ ID NO.3: 5'-CGGGAACCTCCTGGACTACC-3';

a downstream primer SEQ ID NO.4: 5'-CATGGCTGACGAGATCTGAGTG-3';

the probe SEQ ID NO.5 is FAM-CCGCTTCATCGCAGTG-MGB;

the probe SEQ ID NO.6 is VIC-CAGCAGCACCACGGCGTTCACC-BHQ 1.

Preferably, in the primer probe set, the primer probe set contains HLA-A02 gene and ABL1 housekeeping gene.

Preferably, in the primer probe set, the 5 'end of the fluorescent quantitative PCR probe is modified by FAM or VIC, and the 3' end of the fluorescent quantitative PCR probe is modified by NFQ-MGB or BHQ 1.

The invention also provides a kit using the primer probe set.

Preferably, the kit comprises a positive control solution and a blank control solution; the positive control solution contains plasmid DNA; the blank control is TE buffer solution; adding a carrier RNA solution with the effect of protecting plasmid DNA into the positive control solution, wherein the final concentration of the carrier RNA solution is 10-50 ng/[ mu ] L; and the plasmid DNA contains HLA-A02 gene and ABL1 housekeeping gene fragment sequences.

The invention also provides application of the kit in the aspect of detecting HLA-A02 gene for non-diagnosis purposes.

The invention also provides a method for detecting different HLA-A02 genes by using the kit, which is used for non-disease diagnosis and comprises the following steps:

obtaining genome DNA of a sample to be detected, and diluting the DNA to 1 ng/muL-5 ng/muL;

in the same fluorescence PCR amplification reaction system, mixing the genome DNA of a sample to be detected with HLA-A02 specific primers and HLA-A02 specific probes of a primer probe set with nucleotides shown as SEQ ID No.1 to SEQ ID No.6 according to a determined ratio; performing fluorescence PCR amplification by using the genome DNA of the sample to be detected as a template, and collecting a fluorescence signal;

after the fluorescence signal collection is finished, data results are exported and analyzed:

wherein the primer probe set with the nucleotides shown in SEQ ID NO.1 to SEQ ID NO.6 comprises the following nucleotides:

an upstream primer SEQ ID NO.1: 5'-TCCTCGTCCCCAGGCACT-3';

a downstream primer of SEQ ID NO.2: 5'-GAACTGCGTGTCGTCCACG-3';

an upstream primer SEQ ID NO.3: 5'-CGGGAACCTCCTGGACTACC-3';

a downstream primer SEQ ID NO.4: 5'-CATGGCTGACGAGATCTGAGTG-3';

the probe SEQ ID NO.5 is FAM-CCGCTTCATCGCAGTG-MGB;

the probe SEQ ID NO.6 is VIC-CAGCAGCACCACGGCGTTCACC-BHQ 1.

Preferably, in the detection method, the reaction system in the fluorescent PCR amplification also comprises components of TaqMan FAST advance Master Mix and ultrapure water; the kit comprises 5 mu L of TaqMan FAST advance Master Mix, 400nM of an upstream primer SEQ ID NO.1 and a downstream primer SEQ ID NO.2, 300nM of an upstream primer SEQ ID NO.3 and a downstream primer SEQ ID NO.4, 200nM of a probe SEQ ID NO.5 and a probe SEQ ID NO.6, 1 ng-20 ng of genomic DNA, and the balance of ultrapure water.

Preferably, in the detection method, the reaction conditions of the reaction system in the fluorescent PCR amplification at different stages are as follows:

pretreatment: pretreating for 2 minutes at 50 ℃;

pre-denaturation: pre-denaturation at 95 ℃ for 5 min;

the pre-denaturation stage also requires 10 cycles of reaction conditions:

dropping PCR: first, pre-denaturation at 95 ℃ for 15 seconds; secondly, annealing is carried out for 10 seconds at 65 ℃ and 0.5 ℃ every other cycle; finally, extension at 72 ℃ for 20 seconds;

after the touchdown PCR phase, 30 cycles were also performed according to the following reaction conditions:

first, pre-denaturation at 95 ℃ for 15 seconds;

then, annealing was performed at 60 ℃ for 30 seconds.

From the above description, the present invention has the following advantages compared with the prior art:

1. the kit is realized by a PCR technology based on a TaqMan probe, and has high specificity and detection sensitivity;

2. through reasonable primer and probe collocation, the interaction between primers, between primers and between probes can be effectively avoided, so that the specificity of HLA-A02 gene detection is improved, false positive is greatly reduced, and detection errors are reduced;

3. in the detection of the HLA-A02 gene, a recombinant plasmid DNA sample is provided as a positive control, so that the fluorescence analysis of the HLA-A02 gene is more accurate.

Drawings

FIG. 1 is a process flow chart of a method for detecting HLA-A02 gene using the kit of the present invention;

FIG. 2 is a sample kit for HLA-A02 allele carriers of example 1;

FIG. 3 is a sample kit test image of the HLA-A02 allele non-carrier in example 1;

FIG. 4 is a test chart of the positive control liquid sample kit in example 2;

FIG. 5 is a test chart of the negative control liquid sample kit in example 2.

Detailed Description

The preferred embodiments of the present invention will be described in further detail with reference to the accompanying drawings.

The invention provides a primer probe set for detecting HLA-A02, which is composed of HLA-A02 specific primers and probe combinations with nucleotides shown as SEQ ID NO.1 to SEQ ID NO. 6; wherein, the primer probe group with the nucleotides shown as SEQ ID NO.1 to SEQ ID NO.6 comprises the following parts:

an upstream primer SEQ ID NO.1: 5'-TCCTCGTCCCCAGGCACT-3';

a downstream primer of SEQ ID NO.2: 5'-GAACTGCGTGTCGTCCACG-3';

an upstream primer SEQ ID NO.3: 5'-CGGGAACCTCCTGGACTACC-3';

a downstream primer SEQ ID NO.4: 5'-CATGGCTGACGAGATCTGAGTG-3';

the probe SEQ ID NO.5 is FAM-CCGCTTCATCGCAGTG-MGB;

the probe SEQ ID NO.6 is VIC-CAGCAGCACCACGGCGTTCACC-BHQ 1.

The Tm values of the designed HLA-A02 specific primers are all around 58 ℃, and the primers in the combination can be integrated, so that the generation of dimers among the primers is avoided as much as possible; meanwhile, the HLA-A02 is detected by using MGB probes, so that Tm values are all around 68 ℃, the combination between the probes and other non-specific probe sequences is avoided, and the formation of complex dimers among the probes is also avoided, thereby improving the PCR amplification specificity of DNA in HLA-A02.

Preferably, in one embodiment, the 5' end of the fluorescent quantitative PCR probe in the primer probe set is modified with FAM or VIC to indicate the amplification condition of the corresponding gene (FAM, A02; VIC, ABL 1), and the 3' end of the fluorescent quantitative PCR probe is modified with NFQ-MGB or BHQ1 to quench the fluorescent group at the 5' end in the absence of amplification reaction. The TaqMan PCR technique is one of real-time fluorescent PCR. Compared with the traditional PCR, the method adds a probe with two ends respectively marked with a fluorescent reporter group and a quenching group in a reaction system. When the probe structure is complete, the energy of fluorescence emitted by the fluorescence reporter group is transferred to the quenching group, and the quenching effect is presented. If a target sequence exists in the amplification process, the probe molecules are gradually hydrolyzed and cut off in the amplification process, the fluorescent reporter group and the quenching group are mutually dissociated, the fluorescent resonance energy transfer effect between the fluorescent reporter group and the quenching group is blocked, and the fluorescent reporter group sends out a fluorescent signal. As amplification proceeds, the fluorescent signal exhibits a linear increase as the target fragment is amplified.

Aiming at the primer probe set, the invention also provides a kit for detecting the HLA-A02 allele; the kit comprises the primer probe group, and has the advantages of short detection time and high sensitivity.

The kit further comprises a positive control solution and a blank control solution. The positive control solution contains plasmid DNA and a formula capable of ensuring the positive plasmid stability; adding a carrier RNA solution with the effect of protecting plasmid DNA into the positive control solution, wherein the final concentration of the carrier RNA solution is 10-50 ng/mu L, and the plasmid DNA contains HLA-A02 gene and ABL1 housekeeping gene fragment sequences; the blank was TE buffer. The ABL1 housekeeping gene (house-eating gene) because its expression level is less affected by environmental factors and there is little continuous expression change in almost all tissues in each growth stage of an individual; the advantage of housekeeping genes is that they undergo exactly the same processing procedures as the target genes in the sample to monitor the overall process of sampling, transport, nucleic acid extraction and amplification.

As shown in FIG. 1, the method for detecting HLA-A02 gene using the kit for non-disease diagnosis purposes comprises the steps of:

s1, obtaining genome DNA of a sample to be detected; preferably, the final concentration of the diluted DNA is 1 ng/muL-5 ng/muL;

s2, mixing the genome DNA of the sample to be detected with the HLA-A02 specific primer and the HLA-A02 specific probe of the primer probe set in the kit according to a determined proportion, performing fluorescence PCR amplification by taking the genome DNA in the step S1 as a template, and collecting a fluorescence signal;

s3, after the fluorescence signal collection in the step S2 is finished, the data result is derived from the Software 7500 Software v2.3 for analysis, and whether the genome DNA of the detection sample carries the HLA-A02 gene or not is judged.

In step S2, the fluorescence PCR amplification reaction system is 10 μ L total reaction system, including 5 μ L TaqMan FAST advance Master Mix, 400nM each of the upstream primer SEQ ID NO.1 and the downstream primer SEQ ID NO.2, 300nM each of the upstream primer SEQ ID NO.3 and the downstream primer SEQ ID NO.4, 200nM each of the probe SEQ ID NO.5 and the probe SEQ ID NO.6, 1 ng-20 ng genomic DNA, and the rest is filled with water.

Wherein, in a reaction system in the fluorescent PCR amplification, the reaction conditions of different stages are as follows:

pretreatment: pretreating for 2 minutes at 50 ℃;

pre-denaturation: pre-denaturation at 95 ℃ for 5 min;

the pre-denaturation stage also requires 10 cycles to be performed according to the following reaction conditions:

dropping PCR: pre-denaturation at 95 ℃ for 15 seconds, annealing at 65 ℃ (0.5 ℃ reduction every cycle) for 10 seconds, and then extension at 72 ℃ for 20 seconds;

after the touchdown PCR phase, 30 cycles of reaction were performed according to the following reaction conditions:

first, pre-denaturation at 95 ℃ for 15 seconds;

then, annealing was performed at 60 ℃ for 30 seconds.

In the fluorescent PCR amplification reaction system, the HLA-A02 specific primer is used for amplifying a target gene region of a DNA sample, and the HLA-A02 specific probe is combined with the target gene, gradually hydrolyzed during the amplification process and then releases a fluorescent signal. The probe is a linear oligonucleotide, two ends of the probe are respectively marked with a fluorescence reporter group and a fluorescence quenching group, when the probe is complete, a fluorescence signal emitted by the reporter group is absorbed by the quenching group, a fluorescence monitoring system cannot detect the fluorescence signal, and in the PCR amplification process, the probe is cut off, so that the reporter fluorescence group and the quenching fluorescence group are separated, and the fluorescence monitoring system can detect the fluorescence signal.

In addition, the position of the probe is as close to the upstream primer of the corresponding target gene region as possible, in the PCR reaction process, the DNA template in the sample to be detected is denatured into single strands by using high temperature, the temperature is reduced for annealing, and the primer is combined with the DNA template of the target region; meanwhile, the probe can be combined with the DNA template of the target region, and finally, a complementary strand is synthesized along the DNA strand by the primer under the action of polymerase, so that the target region is amplified, an amplification product is continuously extended in the amplification process, and the polymerase hydrolyzes the probe combined with the DNA template to release a corresponding fluorescent signal.

The invention is described in further detail below by way of examples, which are intended to illustrate the invention without limiting it:

example 1:

the genome DNA samples of 542 healthy people are adopted to verify the accuracy of the detection system.

The specific detection method comprises the following steps:

by adopting the PCR reaction system and the reaction conditions described in the specification, 542 healthy human genome DNA samples are selected, the final concentration of the diluted DNA is between 1 ng/muL and 5 ng/muL, and the detection result is compared with the Sanger sequencing result, so that the positive coincidence rate reaches 100 percent, and the negative coincidence rate reaches 100 percent.

FIG. 2 shows the results of amplification curves for samples from HLA-A02 allele carriers; FIG. 3 shows the results of amplification curves for the detection of samples of HLA-A02 alleles from non-carriers. As shown in FIGS. 2 and 3, the expression (i) represents an amplification curve of HLA-A02 gene, and the expressions (ii) and (iii) represent amplification curves of housekeeping gene ABL1 gene.

Therefore, the HLA-A02 allele detection system can specifically amplify the sample of the HLA-A02 allele carrier.

Example 2:

the genome DNA samples, the positive control liquid samples and the negative control liquid samples of 13 healthy people are adopted to confirm the repeatability of the detection system.

The specific detection method comprises the following steps:

by using the PCR reaction system and reaction conditions described in the present specification, 13 samples of healthy human genome DNA, positive control solution and negative control solution, in total, 15 samples of reference samples were selected. The 13 healthy human genome samples comprise 5 HLA-A02 allele carrier samples and 8 HLA-A02 allele non-carrier samples, and the concentration is between 1 ng/. mu.L and 5 ng/. mu.L. And (3) repeatedly detecting 15 reference sample samples for 20 times, dividing the samples into 4 batches, repeatedly detecting each batch for 5 times, comparing the detection result with the Sanger sequencing result, wherein the positive coincidence rate reaches 100%, and the negative coincidence rate reaches 100%.

FIG. 4 shows the result of amplification curve detected by positive control liquid for HLA-A02 allele; FIG. 5 shows the results of amplification curves for HLA-A02 allele negative control liquid samples (actually, there is no corresponding amplification curve in the results); as shown in FIGS. 4 and 5, the symbol (d) represents an HLA-A02 gene amplification curve, and the symbol (d) represents a housekeeping gene ABL1 amplification curve.

The results of the above examples 1 and 2 show that the HLA-A02 allele fluorescent PCR detection system of the invention can accurately and reliably carry out rapid detection on HLA-A02 allele, and compared with other detection methods, the fluorescent PCR quantitative detection system of the invention has higher sensitivity, specificity and accuracy, simple detection procedures, greatly shortened detection time, saved a great deal of manpower, material resources and energy, provides a brand-new rapid and simple HLA-A02 allele detection analysis technology, and has wide clinical application value.

It should be understood that the above description is illustrative of the preferred embodiment of the present invention and is not to be construed as limiting the scope of the invention, which is defined by the appended claims.

Claims (10)

1. A primer probe group for detecting HLA-A02 gene is characterized by comprising HLA-A02 specific primers and HLA-A02 specific probes, wherein the nucleotides of the primers are shown as SEQ ID NO.1 to SEQ ID NO. 6; wherein, the primer probe group with the nucleotides shown as SEQ ID NO.1 to SEQ ID NO.6 comprises the following parts:

an upstream primer SEQ ID NO.1: 5'-TCCTCGTCCCCAGGCACT-3';

a downstream primer of SEQ ID NO.2: 5'-GAACTGCGTGTCGTCCACG-3';

an upstream primer SEQ ID NO.3: 5'-CGGGAACCTCCTGGACTACC-3';

a downstream primer SEQ ID NO.4: 5'-CATGGCTGACGAGATCTGAGTG-3';

the probe SEQ ID NO.5 is FAM-CCGCTTCATCGCAGTG-MGB;

the probe SEQ ID NO.6 is VIC-CAGCAGCACCACGGCGTTCACC-BHQ 1.

2. The primer probe set of claim 1, wherein the primer probe set comprises HLA-A02 gene and ABL1 housekeeping gene.

3. The primer probe set of claim 1, wherein the primer probe set comprises a fluorescent quantitative PCR probe modified with FAM or VIC at the 5 'end and NFQ-MGB or BHQ1 at the 3' end.

4. A kit comprising the primer probe set for detecting HLA-A02 gene according to any one of claims 1 to 3.

5. The kit according to claim 4, further comprising a positive control solution and a blank control solution; wherein the positive control solution contains plasmid DNA; the blank control is TE buffer solution; a carrier RNA solution which plays a role in protecting plasmid DNA is added into the positive control solution, and the final concentration of the carrier RNA solution is 10-50 ng/. mu.L.

6. The kit according to claim 5, wherein the plasmid DNA contains HLA-A02 gene and ABL1 housekeeping gene.

7. A detection method for detecting the aspect of HLA-A02 gene, which is used for non-disease diagnosis purposes, comprises the following steps:

obtaining extracted genome DNA of a sample to be detected;

in the same fluorescence PCR amplification reaction system, mixing the genome DNA of a sample to be detected with HLA-A02 specific primers and HLA-A02 specific probes of a primer probe set with nucleotides shown as SEQ ID No.1 to SEQ ID No.6 according to a determined ratio; performing fluorescence PCR amplification by using the genomic DNA of the sample to be detected as a template, and collecting a fluorescence signal;

after the fluorescence signal collection is finished, deriving a data result, analyzing the data result, and judging whether the genome DNA of the detection sample carries HLA-A02 gene;

wherein, the primer probe group with the nucleotides shown as SEQ ID NO.1 to SEQ ID NO.6 comprises the following parts:

an upstream primer SEQ ID NO.1: 5'-TCCTCGTCCCCAGGCACT-3';

a downstream primer of SEQ ID NO.2: 5'-GAACTGCGTGTCGTCCACG-3';

an upstream primer SEQ ID NO.3: 5'-CGGGAACCTCCTGGACTACC-3';

a downstream primer SEQ ID NO.4: 5'-CATGGCTGACGAGATCTGAGTG-3';

the probe SEQ ID NO.5 is FAM-CCGCTTCATCGCAGTG-MGB;

the probe SEQ ID NO.6 is VIC-CAGCAGCACCACGGCGTTCACC-BHQ 1.

8. The detection method according to claim 7, wherein in the genomic DNA of the sample to be detected, the final concentration of the diluted DNA is 1ng/μ L-5 ng/μ L, and the reaction system for the fluorescence PCR amplification further comprises components of TaqMan FAST advance Master Mix and ultrapure water.

9. The detection method according to claim 8, wherein the fluorescent PCR amplification reaction system is a 10 μ L total reaction system; the kit comprises 5 mu L of TaqMan FAST advance Master Mix, 400nM of an upstream primer SEQ ID NO.1 and a downstream primer SEQ ID NO.2, 300nM of an upstream primer SEQ ID NO.3 and a downstream primer SEQ ID NO.4, 200nM of a probe SEQ ID NO.5 and a probe SEQ ID NO.6, 1 ng-20 ng of genomic DNA, and the balance of ultrapure water.

10. The detection method according to claim 9, wherein the reaction system of the fluorescence PCR amplification has the following reaction conditions in different stages:

pre-denaturation treatment: pretreating for 2 minutes at 50 ℃;

and (3) denaturation treatment: pre-denaturation at 95 ℃ for 5 min;

dropping PCR: pre-denaturation at 95 ℃ for 15 seconds, and annealing at 65 ℃ for 10 seconds by reducing the temperature by 0.5 ℃ every other cycle; then extending for 20 seconds at 72 ℃; continuously executing 10 cycles;

and (3) extension treatment: pre-denaturation at 95 ℃ for 15 seconds, annealing at 60 ℃ for 30 seconds, and collecting fluorescence signals; the execution is continued for 30 cycles.

Images