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Kit and method for warfarin medicine gene mutation site detection

A kit, the technology of warfarin, is applied in the field of kits for the detection of warfarin drug gene mutation sites, which can solve the problems of false positives, high false negatives, harsh PCR conditions, and difficulty in screening for homozygous mutations. High sensitivity, effective typing and detection, and the effect of reducing the number of reaction tubes

Inactive Publication Date: 2016-01-27
SHANG OUTDO BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method is difficult to screen for homozygous mutations, and the false positives and false negatives are high. The PCR conditions are harsh and cannot be typed, and the detection results need to be confirmed by sequencing.
Although gene chips and liquid-phase chips can accurately distinguish specific mutation types, they require high equipment and are difficult to promote on a large scale. They are mostly used in scientific research institutions

Method used

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  • Kit and method for warfarin medicine gene mutation site detection
  • Kit and method for warfarin medicine gene mutation site detection
  • Kit and method for warfarin medicine gene mutation site detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Extraction of Genomic DNA

[0040] The peripheral blood samples of the patients were collected, and the genomic DNA of the samples was extracted with the blood genomic DNA extraction kit of Tiangen Company, and the concentration and purity of the extracted nucleic acids were detected with the NanoDrop1000 nucleic acid and protein analyzer.

Embodiment 2

[0041] Embodiment 2: the design of primer, probe

[0042] Design and screen specific MGB probes that can specifically detect the VKORC1 gene-1639G>ASNP site and 1173C>TSNP site and CYP2C9 gene *2 (430C>T), *3 (1075A>C) SNP sites, above Downstream primers and internal reference primers and probes. The primer probe sequence for detecting the VKORC1 gene-1639G>ASNP site is shown in SEQIDNO:1, SEQIDNO:2, SEQIDNO:3, SEQIDNO:4; the primer probe sequence for detecting the VKORC1 gene 1173C>TSNP site is as shown in SEQIDNO:5 , SEQIDNO:6, SEQIDNO:7, SEQIDNO:8; the primer probe sequence for detecting CYP2C9*2(430C>T) SNP site is shown in SEQIDNO:9, SEQIDNO:10, SEQIDNO:11, SEQIDNO:12 ; Detection of CYP2C9*3 (1075A>C) SNP site primer probe sequence shown in SEQIDNO: 13, SEQIDNO: 14, SEQIDNO: 15, SEQIDNO: 16. The probe sequences of the internal reference primers are shown in SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19.

Embodiment 3

[0043] Embodiment 3: the optimization of reaction system

[0044] (1) Optimization of primer concentration: Under the same conditions in the reaction system, the primer concentration was serially diluted from 200nM to 800nM, and the optimal primer concentration was determined to be 300nM by analyzing and comparing the test results.

[0045] (2) Optimization of the probe concentration: under the same conditions in the reaction system, the probe concentration was serially diluted from 100nM to 300nM, and the optimal probe concentration was determined to be 200nM by analyzing and comparing the test results. .

[0046] (3) Optimization of MgCl2 concentration: Under the same conditions in the reaction system, the MgCl2 concentration was serially diluted from 2mM to 5mM, and the optimal MgCl2 concentration was determined to be 3mM by comparing the test results.

[0047] (4) Optimization of TaqDNA polymerase: various enzymes were added to the reaction system, and the optimal enzyme ...

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Abstract

The invention relates to a kit and method for warfarin medicine gene mutation site detection. The kit comprises PCR hybrid reaction liquid, a primer probe mix, an enzyme mix and a positive control product. Compared with the prior art, the kit has the advantages that the allele specific MGB probe is used to perform amplification detection on SNP sites, and the kit is simple in design, convenient to operate, high in sensitivity and easy in result judgement.

Description

technical field [0001] The invention belongs to the fields of biotechnology and clinical molecular diagnosis, and in particular relates to a kit and a detection method for detecting mutation sites of warfarin drug genes. Background technique [0002] Warfarin is a vitamin K antagonist oral anticoagulant widely used in clinical practice. It produces anticoagulant effect by interfering with the conversion cycle between epoxidized vitamin K and hydroquinone vitamin K. Anticoagulant treatment and prevention of venous thromboembolism, myocardial infarction, ischemic stroke, heart valve replacement, atrial fibrillation, etc. Due to the narrow therapeutic window of warfarin, in the course of clinical use, the stable therapeutic dose varies among different individuals. If the dosage is insufficient, it will lead to thrombosis, and if the dosage is too large, the risk of bleeding will increase, and even life-threatening. Therefore, choosing an appropriate anticoagulant regimen for d...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6858C12Q2535/131C12Q2537/143C12Q2545/101C12Q2521/531
Inventor 吴舒歌郜恒骏段本松张小燕牟晓庆
Owner SHANG OUTDO BIOTECH CO LTD
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