CN114875123A - Standard product for detecting polymorphism of human VKORC1 and CYP2C9 genes and application thereof - Google Patents

Abstract

The invention relates to the field of gene detection, in particular to a standard product for detecting polymorphism of mutated human VKORC1 and CYP2C9 genes and application thereof, wherein homozygous wild and heterozygous mutant cell line DNA containing CYP2C9 genes x 2 and x3 three positions and VKORC1 genes VKORC1-1639 sites is used as a template to perform PCR amplification, and the obtained PCR product is subjected to Sanger sequencing verification to obtain a qualitative standard product of polymorphism of VKORC1 and CYP2C9 genes. The standard substance has good uniformity, and can be used for method verification and quality control of CYP2C19 gene polymorphism detection method.

Description

Standard product for detecting polymorphism of human VKORC1 and CYP2C9 genes and application thereof

Technical Field

The invention relates to the field of gene detection, in particular to a standard product for detecting polymorphism of human VKORC1 and CYP2C9 genes and a preparation method thereof.

Background

Warfarin is the earliest, the most and the most widely used oral anticoagulant drugs in clinic at present, and has been applied to anticoagulant therapy of various diseases, such as primary and secondary prevention of venous thromboembolic diseases (VTE), prevention of atrial fibrillation thromboembolism, valvulopathy, artificial valve replacement, thrombosis in heart cavities and the like, but the clinical curative effect and adverse reaction of warfarin are greatly different from individual to individual, the dosage is difficult to master, and especially in the early stage of the anticoagulant therapy of warfarin, serious bleeding complications are easy to cause. It is estimated that 15.2% of patients taking warfarin develop a blood side effect each year, with fatal major bleeding accounting for 3.5%, and with a difference in warfarin stabilizing dose between individuals of more than ten times. A large number of researches show that the site polymorphism of warfarin target gene VKORC1 gene 1639G & gtA and the site polymorphism of warfarin metabolic enzyme gene CYP2C9 gene are closely related to the anticoagulation effect, and the warfarin dosage difference required by different genotype patients is obvious. The polymorphism of human VKORC1 gene VKORC1-1639G & gtA locus and CYP2C9 gene CYP2C9 x 1, CYP2C9 x 2 and CYP2C9 x3 locus is qualitatively detected, and the dosage and toxicity reaction of warfarin medicine are predicted.

The main methods for detecting gene polymorphism include: restriction Fragment Length Polymorphism (RFLP) detection, biological mass spectrometry detection, sequencing method detection, Taqman probe detection, gene chip detection and the like. These methods, however, differ greatly in their specificity and sensitivity. Compared with common PCR, the current real-time fluorescent quantitative PCR detection method has higher sensitivity and specificity and is widely applied. The project standard can run through the whole process of gene detection based on fluorescence quantitative PCR, and establish quality control of the processes of nucleic acid extraction, PCR amplification and the like. The method has great significance for detecting the out-of-control condition and the out-of-control reason of the results in the laboratory and knowing the difference of the detection results among different medical units.

Disclosure of Invention

The invention aims to provide a standard product for detecting polymorphism of human VKORC1 and CYP2C9 genes and application thereof.

The invention establishes the standard product for detecting the polymorphism of human VKORC1 and CYP2C9 genes for the first time and the application thereof, thereby achieving the aim of the invention.

The technical scheme of the invention is as follows:

(1) extracting genome DNA of a cell line containing CYP2C9 gene x 2 and x3 three positions and homozygous wild and heterozygous mutant type homozygous mutant types at VKORC1 gene VKORC1-1639 sites as a template, and verifying the polymorphic sites by Sanger sequencing;

(2) quantitative determination of CYP2C9 gene x 2, three-position x3 and VKORC1 gene VKORC1-1639 site homozygous wild and heterozygous mutant type cell line genome DNA of homozygous mutant type;

(3) the measurement of the quantity value is carried out by measuring the absorbance value of the nucleic acid of a standard substance, a fluorescence PCR method and a Sanger sequencing method and by adopting a plurality of persons and methods in an experiment. And (3) performing genotyping verification and standard product uniformity verification by adopting a fluorescent quantitative PCR method.

The specific method comprises the following steps:

confirmation was performed by Sanger sequencing using the genomic DNA of the cell line of each of the above-mentioned genotype loci as a template. The sequencing results are shown in Table 1. The purity of the extracted DNA is shown in Table 2.

TABLE 1 Sanger sequencing verification results

TABLE 2 purity of extracted DNA

Sample(s)	Concentration of nucleic acid	OD260/OD280
			GM16654	12.03	1.89
GM12273	13.25	1.87
			GM17222	12.03	1.73
GM17289	13.11	1.81
			GM17204	12.64	1.84
GM17247	12.45	1.94

The principle and the beneficial effects of the invention are as follows:

the human VKORC1 and CYP2C9 gene polymorphism detection standard substance is provided, a cell line quality control substance is close to a clinical sample, the clinical detection is better simulated, the method is superior to the standard substance of the existing method, recombinant plasmids cannot be suitable for all kits on the market due to sequence selection difference among different laboratories, and all individualized medication gene detection kits on the market cannot be accurately identified.

The fixed value precision is good.

Drawings

FIG. 1 is a graph of the sequence of CYP2C9 x 1/' 1 type on CYP2C9 x 2 site by Sanger sequencing.

Figure 2 is a graph of Sanger sequencing for CYP2C9 x 2 site CYP2C9 x 1/' 2 type.

Figure 3 is a graph of Sanger sequencing of CYP2C9 x 2 locus CYP2C9 x 2/' 2 type.

FIG. 4 is a graph of the sequence of CYP2C9 x 1/' 1 type on CYP2C9 x3 site by Sanger sequencing.

FIG. 5 is a graph of the sequence of CYP2C9 x 1/' 3 type on CYP2C9 x3 site by Sanger sequencing.

FIG. 6 is a graph of the sequence of CYP2C9 x 3/' type 3 at the CYP2C9 x3 site by Sanger sequencing.

FIG. 7 is a graph showing Sanger sequencing of VKORC1 sites VKORC1-1639GG type.

FIG. 8 is a graph of Sanger sequencing of VKORC1 site VKORC1-1639GA types.

FIG. 9 is a chart of Sanger sequencing for VKORC1 site VKORC1-1639AA type.

Detailed Description

The following is further detailed by way of specific embodiments:

example 1 preparation of Standard polymorphism of VKORC1 and CYP2C9 genes

1. Cell lines and culture media

Cell lines GM16654, GM12273, GM17222, GM17289, GM17204, GM17247 were purchased from karel cell bank (Coriell Institute), usa, and the required media and culture conditions were as described in the specification. The culture medium is 1640+ 10% FBS, 5% CO ₂ Culturing at 37 ℃.

DNA extraction and Sanger sequencing validation

The genome DNA of 6 cell lines was extracted using a genome purification kit from Baishi medical science and technology, Inc., Jiangsu, and the genome DNA was verified by Sanger sequencing.

3. Genomic purity validation and quantification

The absorbance values of the genome at 260nm, 280nm and 230nm were measured by a NanoDrop method micro-UV spectrophotometer, and Blank was performed using TE as a Blank, and then 2. mu.L of the sample was taken for measurement. And calculating the copy number of the genome according to the concentration to quantify the standard.

4. Genotyping verification by fluorescent quantitative PCR method

And (3) performing PCR amplification by using the genome DNA of the 6 cell lines in the step 2 as a template. 25 μ L of PCR amplification system, the components are shown in tables 3-5 below. The amplification procedure is shown in Table 6.

TABLE 3 CYP2C9 × 2 reaction solution composition Table

TABLE 4 CYP2C9 × 3 reaction solution composition Table

Composition of	Specification of	Dosage of
			MasterBuffer	2×	12.5μL
Upstream primer	10μM	1.0μL
			Downstream primer	10μM	1.0μL
Internal reference upstream primer	10μM	1.0μL
			Internal reference downstream primer	10μM	1.0μL
FAM probe	10μM	0.3μL
			HEX probe	10μM	0.5μL
Enzyme mixture		0.5μL
			DEPC water		Make up to 20 mu L

TABLE 5 composition of VKORC1 reaction solution

Composition of	Specification of	Dosage of
			MasterBuffer	2×	12.5μL
Upstream primer	10μM	1.0μL
			Downstream primer	10μM	1.0μL
Internal reference upstream primer	10μM	1.0μL
			Internal referenceDownstream primer	10μM	1.0μL
FAM probe	10μM	0.3μL
			HEX probe	10μM	0.5μL
Enzyme mixture	-	0.5μL
			DEPC water		Make up to 20 mu L

TABLE 6 amplification procedure

5. F test is carried out on the test result in the uniformity test

Samples were randomly selected from 15 tubes and sampled three times per tube, thus totaling 45 samples. The sampling amount was 200. mu.L/time, DNA was extracted with a human whole blood genome DNA extraction kit (Jiangsu Baishi Nuo medical science and technology Co., Ltd.), and then analyzed by a method of measuring the absorbance value of nucleic acid, to examine the uniformity of the sample in and between bottles.

The intra-cell variance is calculated as follows:

the calculation formula of the inter-cell variance is as follows:

the calculation statistic F is calculated as follows:

second, result in

DNA extraction and Sanger sequencing verification results

The sequencing and identification results of CYP2C9 gene x 2 and x3 three positions and VKORC1 gene VKORC1-1639 site homozygous wild and heterozygous mutant type homozygous mutant Sanger are shown in figures 1-9. The detection result is the corresponding genotype.

2. Genome purity validation and quantification results

The extracted genome is between 1.7 and 2.0 in OD260/OD 280. The statistics of the result of the fixed value are shown in a table 7, the fixed value is carried out by different people, each person measures 5 tubes, each tube repeatedly measures 3 times, all original data are summarized, and finally the total average value of the common measured data after mathematical statistics is 11.90 ng/mul is taken as a standard value. The uncertainty caused during the standard substance calibration was calculated to be 0.19.

TABLE 7 statistics of the results of the valuing

3. Genotyping verification by fluorescent quantitative PCR method

The numbers of CYP2C9 gene x 2 and x3 and VKORC1 gene VKORC1-1639 site homozygous wild and heterozygous mutant type homozygous mutant standard samples are shown in the following table, qualitative detection is carried out by adopting different fluorescent quantitative PCR instrument methods, the CV value is less than 5.0%, and the results are shown in the following tables 8-10.

TABLE 8 ABI7500 fluorescent PCR instrument test results

TABLE 9Roche480 fluorescent PCR Instrument detection results

TABLE 10 MX3000P fluorescent PCR instrument test results

4. For uniformity test result

The in-bottle and inter-bottle uniformity was analyzed by ANOVA and the uniformity test results are shown in Table 11. The homogeneity variance test result shows that the statistic F is less than F0.05, (m-1), m (n-1) samples have no significant difference between bottles, and the samples are uniform.

TABLE 11 results of uniformity test

Claims (3)

1. Each method for preparing a VKORC1 and CYP2C9 gene polymorphism detection standard product comprises the following steps:

(1) extracting the genome DNA of a cell line containing CYP2C9 gene x 2 and x3 three positions and VKORC1 gene VKORC1-1639 site homozygous wild and heterozygous mutant type homozygous mutant type as a template, and verifying the polymorphic site by Sanger sequencing;

(2) quantitative determination of CYP2C9 gene x 2, three-position x3 and VKORC1 gene VKORC1-1639 site homozygous wild and heterozygous mutant type cell line genome DNA of homozygous mutant type;

(3) and (3) performing genotyping verification and standard product uniformity verification by adopting a fluorescent quantitative PCR method.

2. The standard for detecting polymorphisms of VKORC1 and CYP2C9 according to claim 1, wherein: in the (1), the selected cell lines are immortalized cell lines GM16654, GM12273, GM17222, GM17289, GM17204 and GM17247 which can be stably passaged.

3. The use of a VKORC1 and CYP2C9 gene polymorphism detection standard according to any one of the preceding claims, wherein: the determination method of the copy number concentration of each genotype gene is an ultraviolet spectrophotometry.

Images