CN114875126A - Standard product for detecting polymorphism of human CYP2C19 gene and preparation method thereof - Google Patents
Standard product for detecting polymorphism of human CYP2C19 gene and preparation method thereof Download PDFInfo
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- CN114875126A CN114875126A CN202111637414.1A CN202111637414A CN114875126A CN 114875126 A CN114875126 A CN 114875126A CN 202111637414 A CN202111637414 A CN 202111637414A CN 114875126 A CN114875126 A CN 114875126A
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
Abstract
The invention relates to the field of gene detection, in particular to a standard product for detecting polymorphism of a mutant human CYP2C19 gene and a preparation method thereof, wherein cell line DNA containing CYP2C19 gene homozygous wild type and heterozygous mutant type at three sites, and homozygous mutant type at sites 2 and 17 is used as a template to carry out PCR amplification, and a PCR product is obtained and subjected to Sanger sequencing verification to obtain a qualitative standard product of the polymorphism of the CYP2C19 gene. The standard substance has good uniformity, and can be used for method verification and quality control of CYP2C19 gene polymorphism detection method.
Description
Technical Field
The invention relates to the field of gene detection, in particular to a standard product for detecting polymorphism of human CYP2C19 gene and a preparation method thereof.
Background
Clopidogrel is mainly used for treating patients after Acute Coronary Syndrome (ACS) and percutaneous coronary artery stent implantation (PCI), and can obviously reduce the fatality rate of ACS non-revascularized patients and the incidence rate of other cardiovascular events.
The clopidogrel can obviously reduce the incidence rate of stent thrombosis and obviously improve the prognosis of patients with coronary heart disease after Percutaneous Coronary Intervention (PCI) operation, and is used as one of important drugs for standard antiplatelet therapy after PCI operation. However, since 4-30% of patients do not achieve the expected antiplatelet effect due to the difference in reactivity between different individuals to Clopidogrel, platelet reactivity is still high after Clopidogrel application, and the phenomenon of low response (CLR) or no response (CNR) to Clopidogrel is called Clopidogrel low response or Clopidogrel no response, and is collectively called Clopidogrel Resistance (CR). Currently, the phenomenon that patients present different response states to Clopidogrel clinically is called Clopidogrel Response Diversity (CRD). The incidence of clopidogrel resistant patient stent thrombosis and Major cardiovascular adverse events (MACE) events is relatively high. Therefore, clopidogrel response diversity has been the focus of clinical attention. The FDA in the united states proposed a "black box warning," suggesting that patients taking clopidogrel need CYP2C19 genotype testing, identifying differences in patient metabolism to clopidogrel, and predicting risk of adverse clinical events.
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 CYP2C19 gene and a preparation method thereof, and the standard product obtained by the method has high accuracy.
The invention establishes a standard product for detecting the polymorphism of the human CYP2C19 gene and a preparation method thereof for the first time, and achieves the aim of the invention.
The technical scheme of the invention is as follows:
(1) extracting cell line genome DNA containing CYP2C19 gene x 2, x3, x 17 site homozygous wild type, heterozygous mutant type and x 2, x 17 site homozygous mutant type as a template, and verifying the polymorphic sites by Sanger sequencing;
(2) quantitative determination of genomic DNA of cell lines homozygous wild type at three loci, heterozygous mutant type and homozygous mutant type at three loci, 2 and 17, CYP2C19 gene;
(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 |
| GM17204 | 12.27 | 1.92 |
| GM16654 | 12.16 | 1.95 |
| GM17289 | 11.78 | 1.95 |
| GM16688 | 11.78 | 1.89 |
| GM17240 | 11.81 | 1.78 |
| GM17248 | 11.43 | 1.72 |
The principle and the beneficial effects of the invention are as follows:
the preparation method of the human CYP2C19 gene polymorphism detection standard substance is provided, the 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, the recombinant plasmid cannot be suitable for all kits in the market due to the sequence selection difference among different laboratories, and all individualized medication gene detection kits in the market cannot be accurately identified.
The fixed value precision is good.
Drawings
FIG. 1 is a graph of the sequence of CYP2C19 x 1/' 1 type on CYP2C19 x 2 site by Sanger sequencing.
FIG. 2 is a graph of the sequence of CYP2C19 x 1/' 1 type on CYP2C19 x3 site by Sanger sequencing.
FIG. 3 is a graph of the sequence of CYP2C19 x 1/' 1 type on site CYP2C19 x 17 by Sanger sequencing.
FIG. 4 is a graph of the sequence of CYP2C19 x 1/' 2 type by Sanger sequencing on CYP2C19 x 2 site.
Figure 5 is a graph of Sanger sequencing of CYP2C19 x 2 locus CYP2C19 x 2/' 2 type.
FIG. 6 is a graph of the sequence of CYP2C19 x 1/' 3 type on CYP2C19 x3 site by Sanger sequencing.
FIG. 7 is a graph of the sequence of CYP2C19 x 1/' 17 type by Sanger sequencing on CYP2C19 x 17 site.
Figure 8 is a graph of Sanger sequencing of CYP2C19 x 17 site CYP2C19 x 17/' 17 type.
Detailed Description
The following is further detailed by way of specific embodiments:
example 1 preparation of CYP2C19 Gene polymorphism Standard
1. Cell lines and culture media
Cell lines GM17204, GM16654, GM17289, GM16688, GM17240, and GM17248 were purchased from the Calel cell Bank (Coriell Institute), and the required media and culture conditions were 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. The PCR amplification system was 25. mu.L, and the composition is shown in tables 3-5 below. The amplification procedure is shown in Table 6.
TABLE 3 CYP2C19 × 2 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 CYP2C19 × 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 CYP2C19 × 17 reaction liquid 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 6 amplification procedure
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 CYP2C19 gene x 2, x3, site 17 homozygous wild type, heterozygous mutant type and site 2, site 17 homozygous mutant type Sanger are shown in figures 1-8. 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 12.41 ng/mul is taken as a standard value. The uncertainty caused during the standard substance calibration was calculated to be 0.66.
TABLE 7 statistics of the results of the valuing
3. Genotyping verification by fluorescent quantitative PCR method
The standard product numbers of CYP2C19 gene x 2, x3, x 17 site homozygous wild type, heterozygous mutant type and x 2, x 17 site homozygous mutant type 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 9 Roche480 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), and m (n-1) samples have no significant difference between bottles and between bottles, and the samples are uniform.
TABLE 11 results of uniformity test
Claims (3)
1. A preparation method of a CYP2C19 gene polymorphism detection standard substance comprises the following steps:
(1) extracting cell line genome DNA containing CYP2C19 gene x 2, x3, x 17 site homozygous wild type, heterozygous mutant type and x 2, x 17 site homozygous mutant type as a template, and verifying the polymorphic sites by Sanger sequencing;
(2) quantitative determination of genomic DNA of cell lines homozygous wild type at three loci, heterozygous mutant type and homozygous mutant type at three loci, 2 and 17, CYP2C19 gene;
(3) and (3) performing genotyping verification and standard product uniformity verification by adopting a fluorescent quantitative PCR method.
2. The standard for detecting polymorphism in CYP2C19 gene according to claim 1, wherein: in the (1), the selected cell lines are immortalized cell lines GM17204, GM16654, GM17289, GM16688, GM17240 and GM17248 which can be stably passaged.
3. The method for producing a standard substance for detecting a polymorphism in a CYP2C19 gene according to any one of claims, wherein: the determination method of the copy number concentration of each genotype gene is ultraviolet spectrophotometry.
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