Kit for rapid detection of polymorphism of Warfarin metabolic enzyme gene by virtue of pyrosequencing method and application of kit

A pyrosequencing method, a gene polymorphism technology, is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc. The effect of small volume and large promotion and application value

Inactive Publication Date: 2016-08-24
CHANGSHA DIAN MEDICAL SCI INSPECTION CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these methods are time-co

Method used

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  • Kit for rapid detection of polymorphism of Warfarin metabolic enzyme gene by virtue of pyrosequencing method and application of kit
  • Kit for rapid detection of polymorphism of Warfarin metabolic enzyme gene by virtue of pyrosequencing method and application of kit
  • Kit for rapid detection of polymorphism of Warfarin metabolic enzyme gene by virtue of pyrosequencing method and application of kit

Examples

Experimental program
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Effect test

Embodiment 1

[0037] Example 1: Reagents.

[0038] (1) DNA extraction reagents:

[0039] Purchased from QIAGEN Company.

[0040] (2) Reaction solution:

[0041] PCR Buffer: purchased from Fermentas, USA;

[0042] Primers SEQ ID NO: 1-6, synthesized by Shanghai Yingjun Biotechnology Co., Ltd.;

[0043] MgCl 2 : purchased from the U.S. Fermentas company;

[0044] 0.2mM dNTPs: purchased from Fermentas, USA;

[0045] 2U / μL Taq DNA polymerase: purchased from Fermentas, USA.

[0046] (3) Reagent for single-strand purification:

[0047] 75% (v / v) ethanol solution: purchased from Hangzhou Changzheng Chemical Reagent Co., Ltd.;

[0048] 0.2mol / L NaOH: purchased from Shanghai Shisi Hewei Chemical Co., Ltd.;

[0049] 10mmol / L Tris-Acetate (pH 7.6): Tris-base was purchased from Sigma Company in the United States, and anhydrous acetic acid was purchased from Hangzhou Chemical Reagent Co., Ltd.;

[0050] Binding buffer: 10mM Tris-HCl (Tris-base was purchased from Sigma, USA; hydrochloric acid was...

Embodiment 2

[0058] Embodiment 2: detection method.

[0059] Instruments: Bio-Rad S1000 PCR instrument, Beckman Microfuge 22R desktop micro-refrigerated centrifuge, QIAGEN PyroMark Q96 ID sequencer.

[0060] (1) Extract DNA from whole blood samples:

[0061] Referring to the published literature, the corresponding commercial DNA extraction kit was used, and the genomic DNA in the blood was prepared according to the kit instructions, and used as a PCR reaction template for future use.

[0062] After the DNA is dissolved, use a Nano-Space ultraviolet spectrophotometer to measure the nucleic acid concentration. If the nucleic acid concentration is greater than 50ng / ul, it is considered qualified. Store the nucleic acid specimen in a 4°C refrigerator;

[0063] (2) using the DNA obtained in step (1) as a template, and using specific primers to perform PCR amplification;

[0064] Among them, PCR amplification adopts 50μl system, in which DNA template 2μL, 10×PCR buffer 5μL, MgCl 2 (25mmol / L) ...

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Abstract

The invention discloses a kit for rapid detection of polymorphism of a Warfarin metabolic enzyme gene by virtue of a pyrosequencing method. The kit consists of three pairs of specific amplification primers and three sequencing primers; the three pairs of specific amplification primers include two genes, namely a CYP2CP gene and a VOKRC1 gene, wherein the CYP2CP gene contains two pairs of primers; the three sequencing primers specifically relate to three single nucleotide polymorphisms, namely CYP2C9*2, CYP2C9*3 and VKORC1 (-1693); and the nucleotide sequences are as shown in SEQ ID NO.1-9. The detection method disclosed by the invention is not only high in sensitivity, but also direct in result, and simpler, rapider and more accurate in interpretation; the kit is capable of achieving accurate, rapid and high-throughput detection of the Warfarin metabolic enzyme gene; and the kit is relatively high in popularization and application values in personalized medicine.

Description

technical field [0001] The invention belongs to the technical field of molecular diagnosis, and in particular relates to a kit for rapidly detecting gene polymorphisms of warfarin metabolic enzymes by pyrosequencing and an application thereof. Background technique [0002] Warfarin is a coumarin-based oral anticoagulant commonly used in clinical treatment of thrombotic diseases, which has dual effects of anticoagulation and thrombolysis. The anticoagulant therapeutic index range of warfarin is narrow. If the dose is insufficient, the therapeutic effect will not be achieved, and if the dose is slightly excessive, it will cause bleeding, and in severe cases, it will endanger the patient's life. However, there are obvious individual differences and racial differences in the plasma drug concentration and efficacy of warfarin. Studies have shown that the dose of anticoagulant warfarin used by different patients can vary by 20 times, and the maintenance dose of Asians is higher th...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6869C12Q2531/113C12Q2535/122C12Q2565/301
Inventor 任绪义虞闰六施宏
Owner CHANGSHA DIAN MEDICAL SCI INSPECTION CO LTD
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