CN100338230C - Method and detergent for predicting type 2 diabetes susceptibility using PON2 gene the nineth extron polymorphism - Google Patents

Abstract

The invention discloses a method and a reagent for predicting the susceptibility to type 2 diabetes by using the polymorphism of the ninth exon of PON2 gene. By extracting the genomic DNA of the host cell, the SEQ ID of the ninth exon of the PON2 gene of the subject is determined NO: The genotype of the 105th single nucleotide polymorphism site (SNP) in the sequence shown in 1 predicts the susceptibility of the subject to type 2 diabetes: when the 105th genotype is C/C, The susceptibility of the subject is low; when the 105th genotype is C/G or G/G, the susceptibility of the subject is high. The advantages of the present invention are: for the first time, the correlation between the single nucleotide polymorphism site at position 26 of Exon 9 of the PON2 gene and T2DM is clarified, and a method for predicting the susceptibility of T2DM is provided, which can be used for The prevention, auxiliary diagnosis and treatment of T2DM can also be used in the development of new drugs.

Description

A method and reagent for detecting the polymorphism of the ninth exon of PON2 gene

technical field

The invention relates to a method and a reagent for predicting susceptibility to type 2 diabetes by using the ninth exon polymorphism of PON2 gene. The method can be used for auxiliary diagnosis, treatment and new drug development of diseases, and belongs to the field of biotechnology.

Background technique

Diabetes is a chronic disease that cannot be cured temporarily, and can cause a variety of complications, involving the eyes, kidneys, nerves, heart, blood vessels and other tissues and organs. Diabetic patients are often accompanied by disorders of blood lipid metabolism, high blood pressure, obesity and other diseases, and their risk of coronary heart disease and cerebrovascular disease is also significantly higher than that of non-diabetic people. Diabetes has become one of the major diseases that threaten human health, and the economic burden it brings is also increasing day by day. At present, diabetes mellitus (DM) is not a simple disease, but a syndrome of endocrine and metabolic abnormalities, a chronic hyperglycemia state caused by the combination of genetic factors and acquired environmental factors. According to the classification of diabetes recommended by the World Health Organization (WHO) in 1999, it is currently divided into four types, namely type 1 diabetes, type 2 diabetes, gestational diabetes and other special types of diabetes, of which nearly 90% are type 2 diabetes. Type 2 diabetes is more common in middle-aged and elderly people over 40 years old, but it can also occur at any other age. It was once called non-insulin-dependent diabetes mellitus (NIDDM). Its onset has a strong genetic susceptibility, and the incidence increases with the increase of age and weight, and the decrease of exercise. Obesity, especially central obesity, is an obvious predisposing factor for this type of diabetes, which is easy to cause insulin resistance (IR) and accompanied by There is a relative deficiency of insulin secretion. With the development of social economy and the change of people's lifestyle, the incidence of diabetes is increasing year by year.

Studies have shown that type 2 diabetes can be caused by more than one gene abnormality and the positive or negative interaction between pathogenic environmental factors and genes. Genetic factors play a very important role in the pathogenesis of type 2 diabetes. There are two main inheritance modes of type 2 diabetes: (1) monogenic mode, which is caused by mutation of a single gene, but the mutation site is located at a key site of the gene, and its mutation seriously affects the function of the gene; the phenotype of this type of disease is often relatively Serious, such as MODY, etc., this pattern is relatively rare in T2DM patients, accounting for only 5% of the incidence. (2) In the polygenic model, the effect of a single or a few gene mutations is not enough to cause the emergence of an obvious phenotype of the disease, and the disease phenotype appears only when the cumulative effect of multiple gene mutations reaches a certain threshold; and there may be different gene variations Combinations will cause different phenotypic changes; different populations also have different combinations of susceptibility loci. The susceptibility gene combination of T2DM in northern Chinese is still unclear. However, people divide these mutation gene combinations into two types: 1) main effect gene action mode, that is, 1-2 main effect genes play a leading role in disease susceptibility, and the frequency of gene variation in the population is relatively high, and the rest Minor genes of 2 have a small contribution to type 2 diabetes susceptibility. There is no definitive evidence for this pattern in T2DM studies; it has been speculated that this pattern may exist in patients with onset before the age of 30. 2) The mode of action of minor genes, that is, most of the risk alleles originating from multiple loci have a low frequency of occurrence in the population, there is an interaction between them, and the effects are additive, which is consistent with the dosage of quantitative traits -Effect relationship, their joint contribution to T2DM genetic susceptibility leads to critical thresholds for disease development in susceptible populations.

Type 2 diabetes is considered a complex genetic disease, that is, a polygenic disease, which is not only affected by gene-gene interactions but also modified by environmental factors. In recent years, great success has been achieved in identifying susceptibility genes for type 2 diabetes. So that people have a deeper understanding of the pathogenesis of type 2 diabetes. At present, there are two methods for finding susceptibility genes of polygenic genetic diseases: candidate gene screening method and genome-wide scanning positional cloning method. Candidate gene method refers to selecting genes encoding proteins related to blood sugar regulation or energy metabolism as research objects, assuming that their genetic variation is related to genetic susceptibility to type 2 diabetes, and confirming the relationship between these genes and Correlation of susceptibility to type 2 diabetes is a straightforward approach. This approach is relatively easy if the function of the candidate gene is known.

The PON2 gene and PON1 and PON3 genes are located at 7q21-22 and belong to the same gene family. At this gene location, Prochazka et al. found a susceptibility gene for type 2 diabetes in a genome scan study of Pima Indians. PON2 has antioxidant function, can decompose aromatic lipid compounds, and has a certain effect on liver detoxification. The variation of PON2 gene mainly includes Ser 311/Cys discovered by Prochazka et al. and Ala148/Gly discovered by Mochizuki et al. In a population study, Hegele et al. found that homozygosity of the Gly148 allele was associated with fasting hyperglycemia and type 2 diabetes in the Oji-Cree population; found that the PON2 gene C311S was associated with ischemic shock in patients with type 2 diabetes in southern China. In addition, more studies have shown that PON gene family polymorphisms are associated with cardiovascular diseases. These all indicated that the variation of PON2 gene may have a potential association with the occurrence of type 2 diabetes, and it is a valuable candidate gene.

In short, the above background shows that type 2 diabetes is a complex metabolic disease affected by both genetics and environment, and the key functional genes that have an important impact on the susceptibility to type 2 diabetes can be found and used for type 2 diabetes by candidate gene method prediction and prevention. Aromatic esterase 2 (Paraoxonase 2, PON2) is a highly conserved biological enzyme. Researchers believe that it is closely related to the detoxification function of the liver and the antioxidant process of the organism. (Pharmacogenetics 2003 May 13(5): 291-5)

After searching the existing domestic and foreign literature, there is no report on the relationship between the polymorphism of exon 9 of PON2 gene and the susceptibility to type 2 diabetes.

Contents of the invention

The main purpose of the present invention is to provide a method and a reagent for predicting susceptibility to type 2 diabetes by using the ninth exon of PON2 gene.

The second object of the present invention is to provide a reagent for predicting type 2 diabetes, including PCR primers and a kit containing the primers.

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

A method for predicting the susceptibility of type 2 diabetes, by extracting the genomic DNA of the host cell, and determining the single nucleotide polymorphism at position 105 of the sequence shown in SEQ ID NO: 1 of exon 9 of the PON2 gene of the subject The genotype of the site (SNP) predicts the susceptibility of the subject to type 2 diabetes: when the 105th genotype is C/C, the subject's susceptibility is low; the 105th genotype is C /G or G/G, the subject's susceptibility is higher.

Among them, single nucleotide polymorphism (SNP) mainly refers to the DNA sequence polymorphism caused by the variation of a single nucleotide at the genome level. It is the most common type of human heritable variation, and it is the third-generation molecular marker, occupying an absolute predominance among all known polymorphisms.

Genotyping and allele frequency distribution refer to the analysis of the nucleotide sequence sequence of the same allele in the same biological population. Here, it means the determination of the composition of the same SNP site in different individuals, which is genotyping. Allele frequency refers to the proportion of expected alleles in the same type of alleles studied, that is, the proportion of variant alleles.

Disease susceptibility (type 2 diabetes susceptibility) refers to the susceptibility or possibility of a population to a disease.

Case-control association analysis refers to a retrospective study of cause and effect. It first determines the investigation object according to the disease state, divides it into case and control group, and obtains the information about the association between the mark and the disease by comparing the frequency difference of the genetic marker studied between the case group and the control group.

After extensive and in-depth research, the inventors have discovered the 311th Cys(C)→Ser(S) polymorphism in the protein sequence encoded by the ninth exon of the PON2 gene (i.e. the 26th position on the ninth exon The C→G polymorphism exists in the Chinese population, and the present invention is completed on the basis of this.

According to the results of sequencing the PON2 gene of people with type 2 diabetes, it was found that the SNP exists in the Chinese population, and the SNP leads to the change of the 311th amino acid from Cys(C)→Ser(S). Since this is a substitution of amino acids with different properties, it will lead to changes in the activity of the protein encoded by exon 9 of the PON2 gene.

The invention provides an isolated nucleic acid, which has the base sequence shown in SEQ ID NO.1, and the 105th position is G. The nucleic acid sequence is the ninth exon of the PON2 gene, and the amino acid coding region is the 80th-238th position. The complete genome sequence and mRNA and protein sequences of PON2 gene can be obtained from Genebank. The number of the genome sequence in Genebank is gi:27658001.

Those skilled in the art are well aware that there are many analysis methods that can be used to detect whether there is a single nucleotide polymorphism at the site in exon 9 of the PON2 gene. These techniques include (but are not limited to): DNA sequencing, hybridization sequencing, DNA chip, PCR-RFLP, PCR-SSCP, enzymatic mismatch cleavage, denaturing gradient gel electrophoresis, denaturing high performance liquid chromatography (DHPLC), etc.

The primers used in the method or detection kit of the present invention are designed according to the genome sequence of the ninth exon of PON2 gene and its 5' end intron and 3' end non-coding region, and can be synthesized by conventional synthesis techniques. The present invention provides a set of allele-specific primers, which have the base sequences shown in SEQ ID NO.3 and SEQ ID NO.4, the length of which is 18-26bp, and specifically hybridize and amplify the The amplified product of the 105th single nucleotide polymorphism in the sequence shown in SEQ ID NO.1 of the ninth exon of the human PON2 gene.

The test sample used in the present invention is not particularly limited, and for the detection of SNP, it may be genomic DNA extracted from blood, tissue and other samples. For detecting the activity of exon 9 of PON2 gene, any sample expressing exon 9 of PON2 gene can be used, such as blood, liver tissue homogenate and the like.

On the other hand, the detection method of the present invention is used to assess the susceptibility of individuals to suffer from type 2 diabetes and other diseases related to exon 9 of PON2 gene.

The present invention provides a diagnostic kit for detecting T2DM susceptibility, which contains the primer pair for specifically amplifying the ninth exon sequence of PON2 gene of the present invention and the conventional components, reagents, Buffer, etc., those conventional components and detection methods are well known to those skilled in the art. When comparing the amplified product with the SNP at position 26 of exon 9 of the normal PON2 gene, the required chemical reagents also include specific endonucleases. All components, contents, sources and methods of use in the kit of the present invention are as follows:

The kit of the present invention is used for the detection of 10 persons,

Its components, contents and sources include:

20ul 10×PCR buffer (Pharmacia),

4ul 10mM dNTP mixture (Pharmacia),

2ul (5unit/ul) Taq DNA polymerase (Takara),

Each 10ul (10pmol/ul) F1 (SEQ ID NO.2) and R1 (SEQ ID NO.3) primers (self-made),

1.5ml pure water (homemade),

10ul 10× restriction enzyme reaction buffer (Biolab),

4ul (5unit/ul) restriction enzyme DdeI (Biolab).

Instructions:

1) Amplify the ninth exon gene of PON2 gene by PCR: prepare a mixed solution, add 100ng of genomic DNA solution, 2ul 10×PCR buffer, 0.4ul 10mM dNTP, 1.0 unit Taq DNA polymerase and 10pmol F1 and R1 respectively Sense primer and antisense primer, each primer described in Example 2. Next, pure water was added to make the total volume 20ul. The reaction was carried out 35 cycles at 94°C for 0.5 minutes, 58°C for 0.5 minutes, and 72°C for 0.5 minutes. After the reaction was completed, 3 ul of each PCR reaction solution was electrophoresed on an 8% polyacrylamide gel for detection. If a fragment of 331 bp was amplified and a single band was obtained, the amplification was successful.

2) Determining the polymorphism of exon 9 of PON2 gene by treating the PCR reaction product with restriction enzymes. Add 1ul of restriction enzyme reaction buffer and 0.4ul of restriction enzyme DdeI to 5ul of the PCR reaction product amplified above, and digest overnight in a 37°C water bath. Afterwards, electrophoresis was performed on an 8% polyacrylamide gel.

3) Polymorphism typing: the fragment was treated with restriction enzyme Dde I, and when the 105th base was the C allele, bands of 128bp, 105bp, 67bp and 31bp appeared. For G allele, bands of 172bp, 128bp and 31bp appear.

When performing the gene diagnosis of the present invention, it is preferable to use a diagnostic reagent for determining the presence of a mutation type according to exon 9 of the PON2 gene. Methods for mutation types of exon genes. A specific reagent is appropriately selected according to the assay method to be used. The characteristics of the reagents constitute the means necessary for determining the type of mutation defined by the PON2 gene, eg DNA fragments and/or as specific restriction enzymes for the determination. Reagents, such as specially prepared primers for the PCR amplification step for the specific amplified fragment containing the mutation point of the ninth exon of the PON2 gene, are not considered as essential components of the diagnostic reagents of the present invention, and they are also Included in the diagnostic reagent of the present invention.

The advantages of the present invention are: the present invention clarifies for the first time the correlation between the SNP at the 26th position of exon 9 of the PON2 gene and T2DM, and provides a method for predicting the susceptibility of T2DM, which can be used for the prevention, auxiliary diagnosis and treatment of T2DM. It 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 can have a deeper understanding of the content of the invention, but not to limit the present invention.

Description of drawings

Figure 1 is the electrophoretic pattern of PON2 gene exon 9 Ser311Cys polymorphism PCR-RFLP genotype, the first lane on the right is Marker.

Detailed ways

The experimental method that does not indicate specific conditions in the following examples is usually according to conventional conditions, such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions described in the manufacture conditions recommended by the manufacturer.

English abbreviations used in the following examples to represent reagents are as follows.

When needed, autoclave (120°C, 20 minutes) to sterilize

EDTA: disodium ethylenediaminetetraacetic acid

SDS: Sodium Dodecyl Sulfate

TE: 10mM Tris-HCl (pH7.5), 1mM EDTA (pH8.0)

10×PCR buffer: 100mM Tris-HCl (pH8.3), 500mM KCl, 15mM magnesium chloride (MgCl2) 0.01% (W/V) gelatin

dNTP: deoxynucleoside triphosphate

Formamide pigments: 95% deionized formamide, 0.05% dextran blue

APS: ammonium persulfate → 10×TBE: Tris (108 g), boric acid (55 g), EDTA2Na (9.3 g), dissolved in pure water to a total volume of 1 liter.

Example 1 Blood sample collection and extraction of genomic DNA

According to the WHO (1999) standard to clearly diagnose the selected cases, they must take 75g of anhydrous glucose dissolved in 300ml of warm water orally after fasting for 8-12 hours, and detect their plasma glucose value ≥ 11.1mmol/L after 2 hours or have passed the county level or above before. A total of 396 unrelated T2DM patients from Heilongjiang and Beijing, with an average age of 50.0 ± 12.7 years, including 191 males, and 319 healthy control volunteers from the same area, with an average age of 45.2 ± 5.7 years old, including 181 males. All subjects were of Han nationality and signed informed consent, and this research was also approved by the Ethics Committee of the unit.

Genomic DNA was prepared from human peripheral blood according to the following method. In the presence of anticoagulant EDTA, 10 ml of human peripheral blood collected was centrifuged at 2500 rpm for 30 minutes to remove serum. A 0.2% NaCl solution was then added to bring the total volume to 50 ml. The solution was shaken gently 5-6 times and allowed to sit on ice for 15 minutes. Thereafter, the precipitate was collected by centrifugation at 2500 rpm for 30 minutes. With 0.2% NaCl solution, further washing was carried out in a similar manner as before. To the precipitate thus obtained, 10 mM Tris-HCl (pH 8.0) and 10 mM EDTA (4 ml) were added to suspend the precipitate. 10% SDS, 25mg/ml proteinase K and 10mg/ml RNaseA were added to the suspension, the addition amounts were 4ml, 16ul and 20ul respectively, then the suspension was mixed upside down and gently. Then, the suspension was incubated overnight at 37°C. After overnight, 4 ml of the phenol/Tris solution was added and the resulting mixture was mixed by inverting the mixture. The aqueous layer was removed by centrifugation at 3000 rpm for 10 minutes. The aqueous layer was mixed with 4 ml of the phenol/chloroform solution, followed by reverse mixing and centrifugation at 3000 rpm for 10 minutes. Remove the aqueous layer. Finally, extract twice with chloroform to obtain the aqueous phase, add 1/10, 3M NaAC (pH5.2), and twice the amount of cold absolute ethanol to it to precipitate the DNA. Wash with 70% ethanol to obtain genomic DNA. The DNA thus obtained, genomic DNA, was dissolved in TE, and the absorbance of the mixture at 260 nm was quantitatively measured. The concentration of the DNA working solution was corrected to 50ng/ul, and stored in a -20°C refrigerator.

Embodiment 2 Identification and determination of SNP

The invention adopts PCR-RFLP and PCR sequencing technology to detect the ninth exon of PON2 gene.

1) The specific primers are as follows:

Upstream primer: 5'-tggaaaacagggcttattgatga-3' (SEQ ID NO: 2)

Downstream primer: 5'-ctgggtcaatgttgctggttaaa-3' (SEQ ID NO: 3)

2) PCR amplification of the No. 9 exon fragment of the PON2 gene: add 100ng of the genomic DNA solution prepared in Example 1, 2ul of 10×PGR buffer, 0.4ul of 10mM dNTP, 1.0 units of Taq DNA polymerase and 10pmol of the above steps to the reaction vessel 1) Each primer described. Add purified water to make a total volume of 20ul. The reaction was carried out 35 cycles at 94°C for 0.5 minutes, 58°C for 0.5 minutes, and 72°C for 0.5 minutes. After the reaction was completed, 3 ul of each PGR reaction solution was electrophoresed on an 8% polyacrylamide gel to obtain a single band, and observed on a BioRad imager using the Gel Doc 2000 program.

3) A 331bp fragment was amplified and sequenced directly. Huada Zhongsheng Technology Development Co., Ltd. was entrusted to carry out the sequence determination work. "Add 1 ul of restriction enzyme reaction buffer and 0.4 ul of restriction enzyme DdeI to 5 ul of the PCR reaction product amplified above, and digest at 37°C overnight. After that, electrophoresis on 8% polyacrylamide gel. Polymorphism Determination: Treat the fragment with restriction enzyme Dde I, when the 105th base is a C allele, bands of 128bp, 105bp, 67bp and 31bp appear. If it is a G allele, bands of 172bp, 128bp and 31bp appear strip."

Example 3 Correlation research of SNP in exon 9 of type 2 diabetes susceptibility gene PON2 gene

Statistical method: use the Pearson chi-square test in SPSS11.0 software to calculate the Hardy-Weinberg equilibrium test of the PON9 gene polymorphism, and calculate the difference in the distribution of allele frequency and genotype frequency in the case-control population, statistically significant The sex level was set at P<0.05. Univariate Logistic regression analysis was used to calculate the risk OR value of T2DM and its 95% confidence interval (CI).

1. Collection of specimens from patients with type 2 diabetes and normal controls in the Han population in northern China

The peripheral blood samples of the type 2 diabetes group were collected from the Department of Endocrinology, Beijing People's Hospital, and the samples of the normal control group were collected from Harbin Medical University. The samples were from the Han nationality in northern my country, mainly in Beijing and Heilongjiang. Diabetes can be sporadic or a family member of diabetes (only one blood-related member in the same family can be selected). The normal control requires no diabetes patients (including type I diabetes) within three generations of the family. The age and age of the control group and the diseased group Gender match. A total of 614 samples were collected, including 295 from the case group and 319 from the control group. Each selected member should sign the informed consent form.

The diagnosis of type 2 diabetes is strictly in accordance with the standards issued by the WHO in 1997, namely: fasting blood glucose concentration ≥ 7.8mmol/L or postprandial and oral 75 grams of glucose 2 hours blood glucose ≥ 11.1mmol/L is diabetes, after taking sugar 2 Hourly glucose concentration <7.8mmol/L is normal. The basic information of each selected member is recorded, including gender, age, height, weight, heart rate, blood pressure, etc. For the case group, indicators such as age of onset, drug use, comorbidities, and family history were added.

2. SNP locus genotype analysis

Use PCR-RFLP for SNP typing: this SNP changes the restriction endonuclease Dde I enzyme cutting site, and can be amplified by PCR first, and the amplified product is digested with endonuclease Dde I, and after electrophoresis, according to the position of the band To judge the genotype, the wild-type GG homozygote will produce two bands after complete digestion, the mutant homozygote will not produce one band after digestion, and the CG heterozygote will be incomplete digestion. Please refer to Figure 1.

3. Case-control association analysis

In the case and the control group, the typed SNP sites of exon 9 of the PON2 gene were in Hardy-Weinberg equilibrium. Chi-square analysis was performed by SPSS statistical software version 10.0, and the distribution of the genotype frequency and allele frequency of the SNP site in exon 9 of the PON2 gene had statistically significant differences between the two groups (for genotype frequency and allele frequency The frequencies are P<0.01, P<0.05), as shown in Table 1.

Table 1 SPSS statistical analysis results of SNP typing in exon 9 of PON2 gene between case group and control group PON2 HWE(P) no Genotype(%) Allele (%) CC CG GG C G T2DMControl 0.0160.057 458316 296(64.6)239(75.6) 154(33.6)67(21.2) 8(1.8)10(3.2) 746(81.4)545(86.2) 170(18.6)87(13.8) χ ² =15.00, P=0.001 (df=2) χ ² =6.21, P=0.013

As can be seen from the above table, the genotype frequency and allele frequency of the SNP site located in the ninth exon of the PON2 gene are significantly different between the case group and the control group, thus suggesting that this SNP site is related to type 2 diabetes, G The allele is the risk allele of type 2 diabetes, which can significantly increase the incidence of type 2 diabetes, with an OR value of 1.43 and a 95% confidence interval of 1.08-1.89. Thus suggesting that the PON2 gene is a new type 2 diabetes susceptibility gene.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes and modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Embodiment 4 detection kit

Prepare a kit for detecting T2DM-related risks, including a primer pair that can amplify the SNP site of exon 9 of the PON2 gene, and its PCR-RFLP corresponding reagents. The components and contents are as follows, and stored at -20°C:

20ul 10×PCR buffer (Pharmacia),

4ul 10mM dNTP mixture (Pharmacia),

2ul (5unit/ul) Taq DNA polymerase (Takara),

Each 10ul (10pmol/ul) F1 (SEQ ID NO.2) and R1 (SEQ ID NO.3) primers (self-made),

1.5ml pure water (homemade),

10ul 10× restriction enzyme reaction buffer (Biolab),

4ul (5unit/ul) restriction enzyme DdeI (Biolab).

Sequence Listing

<110> Beijing Hospital of the Ministry of Health

<120>A method and reagent for predicting the susceptibility to type 2 diabetes using the polymorphism of the ninth exon of PON2 gene

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<170>PatentIn version 3.3

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gactacagtt tatgccaaca atgggtctgt tctccaagga agttctgtag cctcagtgta 180

tgatgggaag ctgctcatag gcactttata ccacagagcc ttgtattgtg aactctaaat 240

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Claims (2)

1. A set of primers for detecting the polymorphism of the ninth exon of the aromatic esterase 2 gene, having the base sequences shown in SEQ ID NO.2 and SEQ ID NO.3 in the sequence table. 2. A test kit for detecting polymorphism of the ninth exon of aromatic esterase 2 (PON2) gene, characterized in that: it is made up of following reagents: 20ul 10X PCR buffer; 4ul 10mM dNTP mixture; 2ul Taq DNA polymerase, the concentration is 5unit/ul; 10ul each of F1 and R1 primers, F1 and R1 primers have the base sequences shown in the sequence table SEQ ID NO.2 and SEQ ID NO.3 respectively, and the concentration is 10pmol/ul; 1.5ml pure water; 10ul 10X restriction enzyme reaction buffer; 4ul restriction enzyme Dde I, the concentration is 5unit/ul; This kit is for 10 people’s test application, and the storage temperature of the kit is -20°C.

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