CN114561459A

CN114561459A - Standard product for detecting polymorphism of human CYP2D6 gene and application thereof

Info

Publication number: CN114561459A
Application number: CN202111629703.7A
Authority: CN
Inventors: 张陆明; 姜柳; 宗荣芳
Original assignee: Jiangsu Bestnovo Medical Technology Co ltd
Current assignee: Jiangsu Bestnovo Medical Technology Co ltd
Priority date: 2021-12-28
Filing date: 2021-12-28
Publication date: 2022-05-31

Abstract

The invention relates to the field of gene detection, in particular to a mutant standard product for detecting polymorphism of human CYP2D6 gene and application thereof, wherein cell line DNA containing homozygous wild, heterozygous mutant and homozygous mutant at site 10 of CYP2D6 gene is used as a template to carry out PCR amplification, and the obtained PCR product is subjected to Sanger sequencing verification to obtain a qualitative standard product of the polymorphism of CYP2D6 gene. The standard substance has good uniformity, and can be used for method verification and quality control of a fluorescent PCR method for detecting CYP2D6 gene polymorphism.

Description

Standard product for detecting polymorphism of human CYP2D6 gene 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 CYP2D6 gene and application thereof.

Background

CYP2D6 is the first cytochrome P450 enzyme controlled by a single gene, which is mainly expressed in liver, CYP2D6 gene mutation causes enzyme activity and quantity difference, which causes obvious individual difference to substrate metabolism, CYP2D6 substrate drugs used in clinic are mainly antiarrhythmic drugs, beta-receptor blockers, anti-melancholy drugs, antipsychotic drugs and the like, and the substrate drugs metabolized by CYP2D6 have great activity difference of enzyme proteins expressed by different alleles due to gene polymorphism, so that patients carrying different alleles of CYP2D6 have obvious gene difference of pharmacokinetic parameters when using the substrate drugs. Such as: nortriptyline CYP2D6 x 10 allele carrier kinetic parameter AUC higher, clearance lower, half-life longer than CYP2D6 x 1 allele carrier; the clearance rate of the atomoxetine CYP2D6 x 10 allele carrier is lower than that of CYP2D6 rapid metabolism by 50%; approximately 80% of the oral dose of metoprolol is metabolized by CYP2D6, and CYP2D6 x 10 significantly slows the in vivo metabolism of metoprolol. CYP2D6 x 10 is a CYP2D6 allele which is carried by Chinese people with high frequency, and the influence of the gene polymorphism on the safety and the effectiveness of the substrate drug in clinical use is gradually considered.

In vitro diagnostic reagents are important for quality control as important tools for prevention, diagnosis, therapy monitoring, prognosis observation, health status evaluation, and genetic disease prediction. Even though the Ministry of health has standardized the clinical gene amplification testing laboratory from 2002 and has made quality assurance demands, some laboratories are still in the array, and have problems of lack of unified standards, indoor quality control and analysis formalization. The standard substance of the in vitro diagnostic reagent plays a very key role in controlling the quality of the in vitro diagnostic reagent product and ensuring the accuracy of a clinical detection result. Due to many restrictive factors, the national standard substance supply of diagnostic reagents at the present stage cannot completely meet the requirements of registration, production and quality control.

Disclosure of Invention

The invention aims to provide a standard product for detecting polymorphism of human CYP2D6 gene and application thereof, and the standard product has high accuracy and good uniformity.

The invention establishes the standard product for detecting the polymorphism of the human CYP2D6 gene and the application thereof for the first time, thereby achieving the aim of the invention.

The technical scheme of the invention is as follows:

(1) extracting homozygous wild and heterozygous mutant type containing CYP2D6 gene 10 locus, taking the genome DNA of a cell line of the homozygous mutant type as a template, and verifying the polymorphic locus by Sanger sequencing;

(2) quantitative determination of genome DNA of cell lines of CYP2D6 gene 10 three-site homozygous wild type, heterozygous mutant type and 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

Cell lines	Site of the body	Genotype(s)	Sequence of
				GM12273	CYP2D6*10	CYP2D61/1	See FIG. 1
GM17240	CYP2D6*10	CYP2D61/10	See FIG. 2
				GM16654	CYP2D6*10	CYP2D610/10	See FIG. 3

TABLE 2 purity of extracted DNA

Sample(s)	Concentration of nucleic acid	OD260/OD280
			GM12273	11.16	1.77
GM17240	10.87	1.75
			GM16654	12.67	1.84

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

the human CYP2D6 gene polymorphism detection standard is provided, the quality control product of a cell line is close to a clinical sample, the clinical detection is better simulated, the method is superior to the standard product of the existing method, the recombinant plasmid cannot be suitable for all kits on the market due to the difference of sequence selection 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 CYP2D6 x 1/' 1 type on CYP2D6 x 10 site by Sanger sequencing.

FIG. 2 is a graph of the sequence of CYP2D6 x 1/' 10 type by Sanger sequencing on CYP2D6 x 10 site.

Figure 3 is a graph of Sanger sequencing of CYP2D6 x 10 site CYP2D6 x 10/' 10 type.

Detailed Description

The following is further detailed by way of specific embodiments:

example 1 preparation of CYP2D6 Gene polymorphism Standard

1. Cell lines and culture media

Cell lines GM12273, GM17240, GM 16654. Cell lines were purchased from the karel cell bank (Coriell Institute) usa, and the required media and culture conditions were performed according to the instructions. The culture medium is 1640+ 10% FBS, 5% CO₂Culturing at 37 ℃.

DNA extraction and Sanger sequencing validation

The genome DNA of 3 cell lines was extracted using the genome purification kit of 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 3 cell lines in the step 2 as a template. The PCR amplification system was 25. mu.L, the composition of which is shown in Table 3 below. The amplification procedure is shown in Table 4.

TABLE 3 CYP2D6 × 10 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 4 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 CYP2D6 × 10 site homozygous wild type, heterozygous mutant type and homozygous mutant type Sanger are shown in figures 1-3. 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 5, different people carry out fixed value, 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 11.73 ng/microliter is taken as a standard value. The uncertainty caused during the standard substance calibration was calculated to be 0.22.

TABLE 5 statistics of the results of the constant values

3. Genotyping verification by fluorescent quantitative PCR method

The numbers of the CYP2D6 gene x 10 three-site homozygous wild, heterozygous mutant and homozygous mutant standard products 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 6-8.

TABLE 6 ABI7500 fluorescent PCR instrument test results

TABLE 7 detection results of Roche480 fluorescent PCR instrument

TABLE 8 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 9. The homogeneity variance test result shows that the statistic F is less than F0.05, (m-1), and m (n-1) samples have no significant difference between bottles and between bottles, and the samples are uniform.

TABLE 9 results of uniformity test

Claims

1. A CYP2D6 gene polymorphism detection standard substance and application thereof comprise the following steps:

(1) extracting cell line genome DNA containing CYP2D6 gene x 10 point homozygous wild type, heterozygous mutant type and homozygous mutant type as a template, and verifying polymorphic sites by Sanger sequencing;

(2) quantitative determination of genome DNA of cell lines of CYP2D6 gene site 10 homozygous wild type, heterozygous mutant type and 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 gene polymorphism at CYP2D6 site x 10 according to claim 1, wherein: in the (1), the selected cell lines are immortalized cell lines GM12273, GM17240 and GM16654 which can be stably passaged.

3. The use of a standard substance for detecting the polymorphism of the CYP2D6 gene x 10 locus gene according to any one of claims, wherein: the determination method of the copy number concentration of each genotype gene is an ultraviolet spectrophotometry.