Warfarin medication gene detection kit and use method thereof

A gene detection and kit technology, applied in the field of warfarin drug gene detection kits, can solve the problems of high sample processing requirements, low specificity, long detection cycle, etc., and achieves short detection cycle, high sensitivity, and easy operation. Effect

Inactive Publication Date: 2020-09-22
重庆浦洛通基因医学研究院有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The present invention aims at the problems existing in the prior art that the detection of CYP2C9*2, CYP2C9*3, CYP4F2*3 and VKORC1 gene polymorphisms requires high sample processing, long detection cycle, complex operation, high cost and low specificity. , providing a warfarin drug gene detection kit and using method

Method used

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  • Warfarin medication gene detection kit and use method thereof
  • Warfarin medication gene detection kit and use method thereof
  • Warfarin medication gene detection kit and use method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Primer and probe combination design and use

[0041] A warfarin medication gene detection kit, including primer pairs, probes and inhibitors for detecting CYP2C9*2, CYP2C9*3, CYP4F2*3 and VKORC1 gene polymorphisms to be detected, PCR reaction solution, Positive quality control and negative quality control; the sites to be tested are rs1799853 site of CYP2C9*2(430C>T) gene, rs1057910 site of CYP2C9*3(1075A>C) gene, CYP4F2*3(1297G>A At least one of the rs2108622 site of the ) gene and the rs9923231 site of the VKORC1 (-1639G>A) gene. The present invention is screened through large-scale experiments, and the preferred primer and probe sequences are as follows:

[0042] 1. Primer and probe combination for identifying CYP2C9*2(430>C):

[0043] R144C-WF: GGAAGAGGAGCATTGAGGGAC (SEQ ID NO. 1)

[0044] R144C-R:ACAACCAGGACTCATAATGAAAG (SEQ ID NO.3)

[0045] R144C-P: FAM-AAAACCAAGGGTGGGTGACCCTACTCCA-TRAMA (SEQ ID NO. 4)

[0046] R144C-WB: GGATGGGGAAGAGGAGCATTGAGGACTG...

Embodiment 2

[0088] Embodiment 2: the preparation method of kit

[0089] 1. Acquisition of positive quality control products

[0090] The method of obtaining the positive quality control product is as follows: according to the CYP2C9*2, CYP2C9*3, CYP4F2*3 and VKORC1 gene sequences published by the NCBI database, construct a synthetic sequence gene fragment of the plasmid, and then insert the fragment into the T vector, using Escherichia coli The DH5α strain was transformed and the plasmid was extracted, and the quality control plasmids were mixed in equal proportions to be the positive quality control product.

[0091] 2. Preparation of negative quality control

[0092] The negative quality control substance adopted in the present invention is DEPC-treated deionized water.

[0093] 3. Configuration of PCR reaction system

[0094]The kit of the present invention is designed with 8 PCR reaction strips, and each reaction strip can complete the detection of 2 people. Tubes 1-4 are composed ...

Embodiment 3

[0102] Embodiment 3: the use of gene detection kit

[0103] The method for using the kit of the present invention will be described in detail below in conjunction with specific drawings.

[0104] 1. Sample collection

[0105] In this embodiment, peripheral blood of 10 patients was collected, numbered 1-10 respectively. Use vacuum blood collection tubes containing EDTA anticoagulant to collect 5ml of blood samples, let stand at room temperature for 30 minutes, centrifuge at 1500-2000rpm for 10 minutes, and collect plasma and blood cells in sterile screw-top plastic tubes.

[0106] 2. Sample nucleic acid extraction

[0107] Nucleic acid extraction was performed on the vacuum blood sample collected in step 1, and a commercial kit was used for nucleic acid extraction; this invention uses the HiPure Blood DNA Mini Kit (article number: D3111) produced by Meiji Biotechnology, and the experimental steps are carried out with reference to the following instructions.

[0108] In a 1.5...

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Abstract

The invention provides a warfarin medication gene detection kit. The warfarin medication gene detection kit comprises a primer pair used for detecting polymorphism of to-be-detected loci of CYP2C9 * 2, CYP2C9 * 3, CYP4F2 * 3 and VKORC1 genes, probes, an inhibitor, a PCR reaction liquid, a positive quality control product and a negative quality control product; and the to-be-detected loci are at least one of an rs1799853 locus of CYP2C9 * 2 (430C>T) gene, an rs1057910 locus of CYP2C9 * 3 (1075A>C) gene, an rs2108622 locus of the CYP4F2 * 3 (1297G>A) gene and an rs9923231 locusof the VKORC1 (-1639G>A) gene. The invention also provides a use method of the warfarin medication gene detection kit. The problems of high sample treatment requirement, long detection period, complex operation, high cost, low specificity and the like during gene polymorphism are solved.

Description

technical field [0001] The invention is applied in the technical field of gene detection, and in particular relates to a warfarin medicine gene detection kit and a use method. Background technique [0002] Warfarin is an oral anticoagulant of dicoumarin derivatives. It exerts an anticoagulant effect by inhibiting the synthesis of coagulation factors II, VII, IX, and X in liver cells by inhibiting vitamin K. It is widely used clinically for thrombosis Disease prevention and treatment. Clinical practice found that among patients taking warfarin, 15.2% had bleeding side effects every year, of which fatal bleeding accounted for 3.5%. Studies have shown that it is mainly due to differences in drug metabolism genes among individuals, which leads to differences in the dosage of warfarin, and the difference between different individuals can reach more than 20 times. [0003] The study found that the metabolism of warfarin is mainly related to CYP2C9*2 (c.430C>T), CYP2C9*3 (c.10...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/6851
CPCC12Q1/6851C12Q1/6883C12Q2600/106C12Q2600/156C12Q2531/113C12Q2545/113C12Q2563/107
Inventor 颜蓉韩勋领罗锋
Owner 重庆浦洛通基因医学研究院有限公司
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