CN106811546A - A kind of reagent for predicting hypertriglyceridemia neurological susceptibility - Google Patents

Abstract

The invention relates to a reagent for predicting the susceptibility of hypertriglyceridemia, which belongs to the field of biotechnology. The use of a reagent for identifying the genotype of the acid reductone dioxygenase (ADI1) gene polymorphism site in the preparation of a reagent for detecting the susceptibility of a subject to hypertriglyceridemia, which includes : (1) determining the genotype of the rs115172984 site of the ADI1 gene in a sample from the subject, and (2) comparing the measured genotype with a reference genotype, wherein the genotype compared with the reference genotype Differences in the genotypes were used to assess susceptibility to hypertriglyceridemia. The advantages of the present invention are: the present invention clarifies for the first time the correlation between the ADI1 gene polymorphism site and hypertriglyceridemia, and provides a method for predicting the susceptibility of hypertriglyceridemia, which can be used for hypertriglyceridemia The prevention, auxiliary diagnosis and treatment of triglycerideemia can also be used in the development of new drugs.

Description

A reagent for predicting susceptibility to hypertriglyceridemia

technical field

The present invention relates to a reagent for predicting the susceptibility of hypertriglyceridemia, more specifically, predicting the subject's sensitivity to hypertriglyceridemia by determining the polymorphism of gene ADI1 related to hypertriglyceridemia The susceptibility of the method can be used for auxiliary diagnosis, treatment and new drug development of diseases, and belongs to the field of biotechnology.

Background technique

Hypertriglyceridemia (hypertriglyceridemia, HTG) is a heterogeneous triglyceride protein synthesis or degradation disorder. Refers to the elevated levels of triglycerides in the blood, including chylomicrons and pre-β-lipoproteins, which are important risk factors for the occurrence of metabolic syndrome-related diseases such as coronary heart disease, hypertension, and diabetes.

Hypertriglyceridemia mainly has the following clinical manifestations:

The consequence of high triglycerides is that it is easy to cause "blood thickening", that is, blood viscosity caused by excessive lipid content in the blood, deposits on the blood vessel wall, and gradually forms small plaques, that is, atherosclerosis. These massive deposits on the blood vessel wall will gradually expand the area and thickness, making the inner diameter of the blood vessel smaller and the blood flow slower. In addition to the interruption of blood flow, the shedding of obstructions can also cause thrombus; the consequences of high triglycerides are serious damage to the human body no matter where it occurs. If it occurs in the heart, it can cause coronary heart disease and myocardial infarction; if it occurs in the brain, it can cause stroke and stroke; if it occurs in the fundus, it can cause vision loss and blindness; if it occurs in the kidney, it can cause renal failure; Improper flow leads to necrosis.

Hypertriglyceridemia is mainly detected through blood biochemical tests.

Normal triglyceride levels: children <100mg/dL (1.13mmol/L), adults <150mg/dL (1.7mmol/L), borderline hypertriglyceridemia: 250-500mg/dL (2.83-5.65mmol /L), definite hypertriglyceridemia: greater than 500mg/dL (5.65mmol/L).

The present invention is mainly aimed at definite hypertriglyceridemia, that is, the serum triglyceride level is higher than 500 mg/dL (5.65 mmol/L).

At present, the research on the association between single nucleotide polymorphism (SNP) and triglyceride levels is mainly used to find genetic factors that can predict individual hypertriglyceridemia.

SNP refers to the DNA sequence polymorphism caused by a single nucleotide variation at the chromosomal genome level. The frequency in the population needs to be >1%. SNPs are biallelic markers. 70.1% of this single base variation is the same Conversion between bases: such as G/A or T/C, 29.1% are transversions between purine and pyrimidine. C (cytosine) is the most variable site in the human genome, because most of it is methylated cytosine, which can be converted to T (thymine) by spontaneous deamination, and SNP contains 80-90 of known polymorphisms %, is the most common genetic variation.

The distribution of SNPs in single genes and across the genome is heterogeneous due to selection pressure for survival. The number of SNPs in the non-coding region of genes is 4 times that of the coding region, and the total number can reach 3 million. SNP is characterized by its high density (1 per 1 kb on average), strong representation (SNPs located inside the gene may directly affect protein structure or expression level), good genetic stability (compared to microsatellite polymorphism), Ease of automatic analysis (since SNPs are mostly biallelic markers in the population, they can be directly typed by "+/- or I/0") and other characteristics have become good genetic markers.

Research in this field has shown that the relationship between the correlation between a specific SNP genotype and a specific disease and the expected susceptibility to the disease is very complicated. Even for different SNP sites on the same gene, there is no way to refer to each other in relation to the correlation or susceptibility to a certain disease, and even the same SNP site has significant or even qualitative differences for different races .

In the present invention, we have studied the acid reductone dioxygenase Acireductone Dioxygenase 1 (ADI1) gene, the ADI1 gene is located at 2p25.3, contains 5 exons, and its encoded product is an intracellular dioxygenase, Regulate a variety of basic biochemical reactions in cells. So far, there are no research results on the association of ADI1 gene with hypertriglyceridemia.

Contents of the invention

In the present invention, we first discovered the relationship between the genotype of the rs115172984T>C polymorphism site of the ADI1 gene and the susceptibility to hypertriglyceridemia. By extracting the genomic DNA of the host cell and determining the genotype of the rs115172984T>C polymorphism site in the first exon region of the ADI1 gene of the subject, the susceptibility of the subject to hypertriglyceridemia can be predicted: ADI1 When the genotype of rs115172984T/C in the first exon region of the gene is GG, the susceptibility of the subject is the lowest; when carrying the A allele, the susceptibility of the subject is increased.

The present invention also provides an isolated nucleic acid, which has the base sequence shown in SEQ ID NO: 1, and its +401 position is the variation site, marked with the letter "Y". The nucleic acid sequence is the flanking sequence of ADI1 rs115172984 T/C site. Figure 1 is a schematic diagram of the structure of the ADI1 gene and its polymorphic variation sites. The figure contains 5 exons, and the rs115172984T/C site is marked in the corresponding position of the first exon in the ADI1 gene map.

The main object of the present invention is to provide a method for predicting susceptibility to hypertriglyceridemia.

A second object of the present invention is to provide a system for predicting susceptibility to hypertriglyceridemia.

The third object of the present invention is to provide a reagent for predicting susceptibility to hypertriglyceridemia, including PCR primers and a kit containing the primers.

To achieve the above object, the present invention adopts the following technical solutions:

Use of a reagent for identifying the genotype of the acid reductor dioxygenase (ADI1) gene polymorphism site in the preparation of a reagent for detecting the susceptibility of a subject to hypertriglyceridemia, which includes : (1) determining the genotype of the rs115172984 site of the ADI1 gene in a sample from the subject, and (2) comparing the measured genotype with a reference genotype, wherein the genotype compared with the reference genotype Differences in the genotypes were used to assess susceptibility to hypertriglyceridemia.

Said samples include bodily fluids.

The body fluids include peripheral blood, serum, plasma, sputum, synovial fluid, aqueous humor, amniotic fluid, breast milk, semen, prostatic fluid, Cowper's fluid, female ejaculation fluid, sweat, excretion, tear fluid, cystic fluid, pleural effusion , ascitic fluid, pericardial fluid, chyle, bile, interstitial fluid, menstrual blood, pus, vomit, vaginal discharge, mucous membrane secretion, pancreatic juice, bronchopulmonary aspirated fluid, blastocoel fluid, or umbilical cord blood.

The reference genotype refers to that the genotype at the rs115172984 site is GG.

A method for evaluating susceptibility to hypertriglyceridemia, receiving data related to genotypes including the rs115172984 locus of the ADI1 gene of a subject, and comparing the genotype of the subject with a reference gene considered to have low susceptibility genotype, as well as a warning signal if the subject's genotype differs from the reference genotype.

The reference genotype means that the genotype at the rs115172984 site is GG, and the reference genotype is the non-risk allele of hypertriglyceridemia.

A system for evaluating susceptibility to hypertriglyceridemia, including a receiver, a database, and a processor; wherein, the receiver is used to receive relevant data including the genotype of the subject's ADI1 gene rs115172984 site, stored in the database There is reference genotype data that is considered to have low susceptibility, and the processor is used to compare the data related to the genotype of the subject's ADI1 gene rs115172984 site with the reference genotype data, and compare the subject's genotype with the reference genotype data. A warning signal is issued when the genotype is different; the reference genotype refers to the genotype at the rs115172984 locus as GG.

A set of primers for evaluating the susceptibility of hypertriglyceridemia is the nucleotide sequence shown in SEQ ID NO: 2 and SEQ ID NO: 3 in the sequence listing, and can specifically amplify the Product at position +401 in the sequence shown by ID NO:1.

The present invention provides a diagnostic kit for detecting susceptibility to hypertriglyceridemia, which contains the primer pair for specifically amplifying the +401 site of the ADI1 gene of the present invention and the conventional components of the kit for PCR amplification detection , reagents, buffers, etc., those conventional components and detection methods are well known to those skilled in the art. All components, contents, sources and methods of use in the kit of the present invention are as follows:

30 μL 10×PCR buffer;

5 μL 10mM dNTP mixture;

5 μL TaqDNA polymerase, the concentration is 2U/μL;

2.5 μL F1 primer, the concentration is 10pM/μL, which is the nucleotide sequence shown in SEQ ID NO: 2 in the sequence table;

2.5 μL R1 primer 10pM/μL, the concentration is 10pM/μL, which is the nucleotide sequence shown in SEQ ID NO: 3 in the sequence table;

235 μL pure water.

Instructions:

(1) PCR amplification: amplify the fragment of the first exon region of the ADI1 gene by PCR, and prepare a mixture: 3 μL of 10×PCR reaction buffer, 0.5 μL of 10 mM/L dNTP, 0.5 μL of TaqDNA polymerase, 10 pM/ L upstream primer 0.5 μL, 10pM/L downstream primer 0.5 μL, genomic DNA 2 μL, add pure water to 30 μL. The PCR reaction conditions were pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 65°C for 30 s, extension at 72°C for 25 s, a total of 35 cycles, and a total extension at 72°C for 2 min. Before PCR, 20 μL of paraffin oil was added to each system to prevent the liquid from volatilizing.

(2) Genotype determination: The PCR product was directly sequenced, and the genotype was determined according to the difference of the fluorescence signal.

The assay method of the present invention measures the genomic DNA derived from humans, and the source of the sample is not limited, such as: body fluid (blood, ascites, urine, etc.), tissue cells (such as liver tissue) and the like. Genomic DNA can be prepared by extracting and purifying these samples. Adjust the concentration of genomic DNA to make it as consistent as possible. Using genomic DNA as a template, nucleic acid fragments containing ADI1 gene mutation sites can be amplified to obtain a large number of samples for determination. The sample obtained by amplifying the DNA fragment containing the ADI1 gene variation point is particularly suitable for use as a determination material.

When gene-assisted diagnosis is performed, the present invention is preferably applicable to an auxiliary diagnostic reagent for determining the presence of a mutation type of the ADI1 gene, and the auxiliary diagnostic reagent includes as an essential component a specific reagent corresponding to the method for determining the mutation type of the ADI1 gene. Appropriate specific reagents, such as DNA fragments and/or primers for PCR amplification steps, are selected according to the assay method employed.

The advantages of the present invention are: the present invention clarifies for the first time the correlation between the ADI1 gene polymorphism site and hypertriglyceridemia, and provides a method for predicting the susceptibility of hypertriglyceridemia, which can be used for hypertriglyceridemia The prevention, auxiliary diagnosis and treatment of triglycerideemia can also be used in the development of new drugs.

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that the public has a deeper understanding of the content of the invention, and is not a limitation of the present invention. All equivalent replacements in the field made according to the disclosure of the present invention belong to this invention. protection scope of the invention.

Description of drawings

Figure 1 is a schematic diagram of the structure of the ADI1 gene and its polymorphic variation sites

Figure 2A is the sequencing result of ADI1 rs115172984 TT genotype

Figure 2B is the ADI1 rs115172984 CT genotype sequencing results

detailed description

The abbreviations used to represent reagents in the following examples are as follows:

10×PCR buffer: 10mM Tris-HCL (pH=8.3), 0.5M potassium chloride (KCL), 10mM magnesium chloride (MgCL), 0.01% (W/V) gelatin

dNTP: deoxynucleoside triphosphate

EDTA: ethylenediaminetetraacetic acid

TE: 10mM Tris-HCl (pH=7.5), 1mM EDTA (pH=8.0)

Example 1 Blood Sample Collection and Genomic DNA Extraction

(1) According to the diagnostic criteria formulated by the Chinese Medical Association, 100 unrelated patients with hypertriglyceridemia and 148 healthy volunteers from the same area were selected. All subjects were of Han nationality and signed written informed consent. This study was approved by the Beijing Hospital of the Ministry of Health and the Ethical Review Committee of the Institute of Gerontology of the Ministry of Health. .

(2) According to the following method, human genomic DNA was prepared. ①First add 1000 μL of erythrocyte lysate to a labeled 1.5mLEP tube, then add 400 μL of LEDTA anticoagulant blood (invert and mix 3-5 times before adding anticoagulant blood), invert and mix well, and let stand at room temperature for 10 minutes; ②Centrifuge at 13000rpm for 30 Seconds later, remove the supernatant; ③Add 480 μL of nucleic acid lysate to the obtained precipitate, flick the tube wall, mix well, add 20 μL of proteinase K (dilute proteinase K 20 times with cleavage solution), invert and mix, 65 ℃ incubator for 10 minutes, (during this period, mix up and down from time to time to ensure no clots); ④ Take it out and cool it down to room temperature, add 300 μL of protein precipitation solution, mix well by inverting, let stand for 10 minutes, and centrifuge at 13000rpm for 2 minutes; Transfer the supernatant to a new EP tube, add 670 μL of pre-cooled isopropanol, and mix thoroughly by inversion (more than 10 times), it can be seen that the linear DNA gradually forms small clumps, centrifuge at 13,000 rpm for 2 minutes; ⑥ Discard the supernatant and Make sure that the precipitate remains in the EP tube, add 670 μL of 70% ethanol, mix up and down, and centrifuge at 13,000 rpm for 2 minutes; ⑦ Discard the supernatant and let the ethanol in the tube evaporate; ⑧ Add TE solution (400 μL), fully dissolve, and extract the genomic DNA Analyze the concentration and purity, draw part of the DNA solution as the working solution, correct the concentration to 20ng/μL, store it at 4°C for use, and store the remaining genomic DNA in a -20°C refrigerator.

Example 2 Identification and identification of variable sites

The invention adopts PCR-sequencing analysis method to detect the genotype of the +401 site (the allelic site is T/C) in the first exon region of the ADI1 gene. Fig. 2 is a sequence diagram of the variation site of ADI1 gene.

1. Determination of PCR-sequencing primers

The DNA base sequence (SEQ ID NO: 1) near rs115172984 T/C was retrieved from Genebank, and the primer design was completed under Oligo7.0 software. The target fragment is located in the first exon region of the ADI1 gene, with a total length of 512bp. The sense strand F1 (+154bp-+175bp) and the antisense strand R1 (+644bp-+665bp) are determined. The specific primer sequences are as follows:

F1: 5'-CTTACACCCCCTCCCCTTTTGA-3' (SEQ ID NO: 2)

R1: 5'-TCCATCTCCTCTTACAAGGCCAC-3' (SEQ ID NO: 3)

2. PCR-sequencing reaction system and conditions

Amplify the partial fragment of exon 1 of ADI1 gene by PCR. The PCR reaction system is: 3 μL of 10×PCR reaction buffer, 0.5 μL of 10mM/L dNTP, 0.5 μL of TaqDNA polymerase, 0.5 μL of 10pM/L upstream primer, 10pM /L downstream primer 0.5 μL, genomic DNA 1 μL, add deionized water to 30 μL. During PCR, 20 μL of paraffin oil was added to each system to prevent the liquid from volatilizing. The PCR reaction conditions were pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 65°C for 30 s, extension at 72°C for 25 s, a total of 35 cycles, and a total extension at 72°C for 2 min.

3. Sequencing to determine genotype

The PCR product was detected by 8% polyacrylamide gel electrophoresis, and the gel imaging system was observed to pass the inspection and then sent to the BGI Sequencing Department for sequencing verification. The results are shown in Figure 2A and Figure 2B.

Example 3 Correlation between gene SNP and hypertriglyceridemia

Statistical methods: The Hardy-Weinberg balance test was used to study the group representativeness of the samples. Using the Pearson chi-square test in SPSS11.0 software to calculate the distribution frequency of alleles and genotypes of the ADI1 gene rs115172984 T/C site between the hypertriglyceridemia case group and the normal control group, hypertriglyceridemia The OR value of the disease risk and its 95% CI confidence interval, with P<0.05 as the standard of significant difference.

Results: The distribution of the genotype and allele frequency of the rs115172984 T/C site on the ADI1 gene located in the 2p25.3 region between the cases and the control group is shown in Table 1.

Table 1: Distribution of genotype and allele frequency of ADI1(rs115172984 T/C) locus between case and control group

Note: OR: odds ratio; CI: confidence interval. *A allele is the risk allele for hypertriglyceridemia. Subjects were divided into hypertriglyceridemia risk allele (GA) carriers and non-risk allele (GG) carriers.

It can be seen from Table 1 that the C allele of ADI1 (rs115172984 T/C site), that is, the G allele on its DNA complementary strand, has a significantly higher distribution frequency in the patient population than in the healthy normal population. The distribution frequency of alleles (0.075vs.0.024) has a significant difference (P=0.006), and the OR value of the A site is 3.347, 95% CI: 1.339-8.366; in the risk of hypertriglyceridemia, etc. Among allele (CT) carriers and non-risk allele (TT) carriers, the distribution frequency of risk genotypes in the case group was significantly higher than that in the control group (P=0.005), both indicating that the ADI1 gene rs115172984 T/ The C locus is positively correlated with hypertriglyceridemia, which may increase the risk of hypertriglyceridemia.

Embodiment 4 detection kit

A kit for detecting risks associated with hypertriglyceridemia is prepared, including a primer pair that can amplify the SNP+401 site of the ADI1 gene, and other PCR-HRM corresponding reagents. The kit of the present invention is used for detection of 10 people, and is stored at -20°C in the dark, and its components, contents and sources include:

30 μL 10×PCR buffer (Pharmacia),

5 μL of 10 mM dNTP mix (Pharmacia),

5 μL Taq DNA polymerase (2 U/μL) (Takara)

2.5 μL F1 primer (SEQ ID NO: 2) (10 pM/μL)

2.5 μL R1 primer (SEQ ID NO: 3) (10 pM/μL),

235 μL pure water.

After PCR-sequencing detection, the rs115172984 T/C polymorphism in the first exon region of the ADI1 gene can be easily detected.

Examples of the utility of the present invention:

The detection method of ADI1 gene polymorphism of the present invention can be used for analyzing the rs1057079 C allele on the ADI1 gene of human autosome 2p25. Auxiliary diagnosis of triglyceridemia and assessment of individual risk of hypertriglyceridemia, in order to facilitate early intervention and treatment of hypertriglyceridemia.

Utilizing the present invention to describe the base variation of ADI1 gene rs115172984 T/C site, as one of the biomarkers, it can be used as a molecular target screening for drug design to help find active molecules that regulate ADI1 expression and promote high triglycerides Research and development of new drugs for esteremia.

The nucleic acid sequence and hypertriglyceridemia-related sites established by the invention for detecting the polymorphism of the ADI1 gene can be applied to a kit for gene-aided diagnosis of hypertriglyceridemia with high sensitivity and specificity.

As mentioned above, it was concluded that the polymorphism of rs115172984 T/C site of ADI1 gene was significantly correlated with hypertriglyceridemia. Therefore, the determination of this polymorphism according to the present invention can be used for gene-assisted diagnosis of hypertriglyceridemia.

The present invention describes the new mutation site associated with ADI1 gene hypertriglyceridemia, and provides a method for determining the polymorphism of the ADI1 gene, and, according to the present invention, only a small amount of DNA sample is enough to determine the polymorphism of the gene attitude. As a result, the present invention provides a gene-aided diagnosis method for determining polymorphisms of genes associated with hypertriglyceridemia.

sequence listing

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Claims (10)

1. The use of a reagent for identifying the polymorphic site genotype of the acid reductone dioxygenase (ADI1) gene in the preparation of a reagent for detecting the susceptibility of the subject to hypertriglyceridemia, It includes: (1) determining the genotype of the rs115172984 site of the ADI1 gene in a sample from the subject, and (2) comparing the determined genotype with a reference genotype, wherein the genotype is compared with the reference genotype The genotype difference in ratio was used to assess susceptibility to hypertriglyceridemia. 2. The use according to claim 1, characterized in that: said sample comprises body fluid. 3. The use according to claim 2, characterized in that: said body fluids include peripheral blood, serum, plasma, sputum, synovial fluid, aqueous humor, amniotic fluid, breast milk, semen, prostatic fluid, Cowper's fluid, female ejaculation fluid, sweat, excretion, tears, cystic fluid, pleural effusion, ascites fluid, pericardial fluid, chyle, bile, interstitial fluid, menstrual blood, pus, vomit, vaginal secretion, mucous membrane secretion, pancreatic juice, bronchopulmonary Aspirated fluid, blastocoel fluid, or cord blood. 4. The use according to any one of claims 1-3, characterized in that: the reference genotype means that the genotype at the rs115172984 site is GG. 5. A method for evaluating susceptibility to hypertriglyceridemia, characterized in that: receiving data related to the genotype including the subject's ADI1 gene rs115172984 site, and comparing the subject's genotype with those considered to have A low susceptibility reference genotype is compared and a warning signal is issued if the subject's genotype differs from the reference genotype. 6. The method according to claim 5, wherein the reference genotype means that the genotype at the rs115172984 site is GG. 7. A system for evaluating susceptibility to hypertriglyceridemia, characterized in that: it includes a receiver, a database and a processor; wherein the receiver is used to receive the genotype of the subject's ADI1 gene rs115172984 site Relevant data, the reference genotype data considered to have low susceptibility is stored in the database, and the processor is used to compare the relevant data of the genotype of the subject's ADI1 gene rs115172984 site with the reference genotype data, and warning signal if the genotype of the patient differs from the reference genotype. 8. The system according to claim 7, wherein the reference genotype means that the genotype at the rs115172984 site is GG. 9. A set of primers for evaluating susceptibility to hypertriglyceridemia, characterized in that it is the nucleotide sequence shown in SEQ ID NO: 2 and SEQ ID NO: 3 in the Sequence Listing. 10. A test kit for evaluating susceptibility to hypertriglyceridemia, characterized in that it consists of the following reagents: 30 μL 10×PCR buffer; 5 μL 10mM dNTP mixture; 5 μL TaqDNA polymerase, the concentration is 2U/μL; 2.5 μL F1 primer, the concentration is 10pM/μL, which is the nucleotide sequence shown in SEQ ID NO: 2 in the sequence table; 2.5 μL R1 primer 10pM/μL, the concentration is 10pM/μL, which is the nucleotide sequence shown in SEQ ID NO: 3 in the sequence table; 235 μL pure water.