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Kit for detecting genotype of gene CYP2C19 at SNP site rs4986893

A technology of CYP2C19 and genotyping, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems of adverse reaction drug dosage, treatment failure, insufficiency, etc., and achieve rapid detection, high sensitivity, and good specificity Effect

Inactive Publication Date: 2017-05-24
SUZHOU BAIYUAN GENT CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to differences in drug metabolism in different individuals, serious adverse reactions or insufficient drug dosage may occur after administration, resulting in treatment failure

Method used

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  • Kit for detecting genotype of gene CYP2C19 at SNP site rs4986893
  • Kit for detecting genotype of gene CYP2C19 at SNP site rs4986893
  • Kit for detecting genotype of gene CYP2C19 at SNP site rs4986893

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 CYP2C19Preparation of rs4986893 standard

[0054] To establish an HRM analysis method, the external standard required by the method must first be prepared. The standard should contain highly conserved and specific sequences, and the high specificity of the reaction must be ensured. The synthesis of the present invention comprises CYP2C19 The wild-type and homozygous mutant DNA sequences of the rs4986893 (c.636G>A) site were cloned into the pMD18-T vector by gene recombination technology to construct the wild-type and homozygous recombinant plasmid pMD18-T-rs4986893 mutant type, and carry out corresponding PCR identification and sequencing identification, and finally quantify it as a standard for the method to be established: the wild-type recombinant plasmid is a recombinant negative plasmid, and the homozygous mutant recombinant plasmid is a recombinant positive plasmid, which is the method for the next step and foundation for assessment.

[0055] 1. Const...

Embodiment 2

[0098] Example 2 HRM-PCR detection CYP2C19 rs4986893 method building

[0099] 1. Preparation of samples to be tested

[0100] The blood genome DNA of 30 samples was extracted for use as CYP2C19 Template for gene PCR amplification. The extraction steps of blood genomic DNA are as follows:

[0101] (1) Take 1ml whole blood, add 3ml TE, invert and mix well, let stand for 10min, centrifuge at 8000rpm for 5min, discard the supernatant;

[0102] (2) Repeat the above steps 2-3 times until the precipitate is white;

[0103] (3) Add 900μl 10% SDS and 10μl 10mg / ml proteinase K, and bathe in water at 55℃ for 1h;

[0104] (4) Cool the centrifuge tube to room temperature, add an equal volume of phenol / chloroform / isoamyl alcohol (25:24:1) to mix, and centrifuge at 12000rpm for 10min;

[0105] (5) Carefully absorb the supernatant, then add an equal volume of phenol / chloroform / isoamyl alcohol (25:24:1) to mix, and centrifuge at 12,000rpm for 10min;

[0106] (6) Take the supernatant, ad...

Embodiment 3

[0155] Example 3 Application of the detection method of the present invention to detect samples CYP2C19 rs4986893 genotype

[0156] Using the detection kit and detection method in the steps of Example 2, HRM analysis was performed on 30 samples, compared with the melting curves of the recombinant positive plasmid and the recombinant negative plasmid, and the mutation type in the sample was determined accordingly, and according to the results of the HRM analysis, target One sample of each genotype was randomly selected for Sanger sequencing verification at Sangon Biotech (Shanghai).

[0157] HRM analysis results are shown in Table 1 and figure 2 , the data show that in 30 samples CYP2C19 Gene rs4986893 was G>A heterozygous mutant in 7 cases, G>A homozygous mutant in 6 cases, and the remaining 16 cases were wild type.

[0158] For Sanger sequencing results, see image 3 : wherein a is the sequencing result of sample number 2, b is the sequencing result of sample number 11, ...

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Abstract

The invention provides a kit for detecting the genotype of a gene CYP2C19 at an SNP site rs4986893. The kit comprises a primer pair and a PCR detection reaction reagent, wherein the primer pair is one of (1) and (2): (1) an upstream primer: 5'-CATCAGGATTGTAAGCACCCCC-3' and a downstream primer: 5'-TTTCTCAGGAAGCAAAAAACTTGG-3'; (2) complementary sequences for the sequences in (1). The kit disclosed by the invention can accurately detect the genotype of the gene CYP2C19 at the SNP site rs4986893, and is high in sensitivity, high in specificity and quick in detection. By the application of the kit, the parting result and the sequencing result of the genotype of the gene CYP2C19 at the SNP site rs4986893 are completely consistent, so that the kit can be used for individualized treatment based on CYP2C19 rs4986893 related medicines.

Description

technical field [0001] The present invention relates to the detection of CYP2C19 Gene SNP site rs4986893 genotype kit. Background technique [0002] It has been confirmed that CYP2C19 not only participates in the metabolism of antiepileptic drugs, but also many clinically important drugs are its substrates. The oxidative metabolism of drugs in humans can be divided into two phenotypes, one is a strong metabolizer and the other is a poor metabolizer. The latter is mainly due to the two defective alleles of CYP2C19*2 and CYP2C19*3. caused. Due to the differences in drug metabolism in different individuals, severe adverse reactions or insufficient drug dosage may occur after administration, resulting in treatment failure. CYP2C19 can catalyze a series of drugs in the body, such as S-mephenytoin (S-mephenytoin, S-MP), omeprazole (omeprazole, OPZ), diazepam (DZ), nordiazepam juice (demethyldiazepam), imipramine (IMI), tolbutamide (D860), phenobarbital (PB), proguanil and chlo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6883C12Q2600/106C12Q2600/156
Inventor 谢小冬车团结王莉尤崇革李琳李亚鹏
Owner SUZHOU BAIYUAN GENT CO LTD
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