Primer system for detecting gene SNP (single nucleotide polymorphism) related to warfarin dosage and application of primer system

A drug dosage and gene technology, applied in the biological field, can solve the problems of low sensitivity, inability to perform multiple detections, and low inherent accuracy

Inactive Publication Date: 2012-09-26
毅新兴业(北京)科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because the method is slow in operation, low in accuracy, easy to be contaminated clinically, and cannot perform multiple detections, it cannot solve the above technical problems
[0011] Chinese patent application 201010234597.8, "a kit for detecting SNP sites of genes related to warfarin individualized drug use and its PCR amplification method", involves PCR combined electrophoresis detection method, which can only detect 2 SNP sites , can not perform multiple detection, and its accuracy is low, and it is easy to be contaminated clinically, so it cannot solve the above technical problems
[0012] Chinese patent application 201110211124.0, "Kit for detecting SNP sites related to warfarin individualized drug use and its multiplex PCR amplification method and detection method", discloses a method for detecting 4 SNP sites by PCR combined with electrophoresis, Compared with the previous technology, this method has made some progress, but there are still a small number of detected SNP sites, and the inherent accuracy of electrophoresis display results is low, and the defects of easy clinical contamination, so it cannot solve the above technical problems
Since this method only involves two SNP sites, and fluorescent qu

Method used

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  • Primer system for detecting gene SNP (single nucleotide polymorphism) related to warfarin dosage and application of primer system
  • Primer system for detecting gene SNP (single nucleotide polymorphism) related to warfarin dosage and application of primer system
  • Primer system for detecting gene SNP (single nucleotide polymorphism) related to warfarin dosage and application of primer system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0129] Example 1: Primer design and synthesis.

[0130] For VKORC1 gene rs9923231 (-1639G / A), CYP2C9 gene rs1799853 (CYP2C9*2), CYP2C9 gene rs1057910 (CYP2C9*3), CYP2C9 gene rs9332131 (CYP2C9*6), CYP4F2 gene rs210862 Point (V433M), GGCX gene rs11676382 site, CALU gene rs339097 site and other 7 gene polymorphism sites related to warfarin dosage, design corresponding specific PCR primer core sequences (SEQ ID No: 1 to SEQ ID No: 14) and specific extension primer core sequences (SEQ ID No: 15 to SEQ ID No: 21).

[0131] Among them, in order to prevent the PCR primers from entering the detection window of the mass spectrometer and interfering with the detection effect, the 5' end of each PCR primer can add a certain number of bases on the basis of the core sequence (SEQ ID No: 1 to SEQ ID No: 14) , such as a 10bp tag (ACGTTGGATG), to increase the molecular weight of the PCR primers, thereby exceeding the detection window of the mass spectrometer.

[0132] Relevant primers were s...

Embodiment 2

[0134] Embodiment 2: sample DNA extraction.

[0135]A total of 10 patients with clinical manifestations of warfarin resistance were collected. Among them, sample collection, DNA extraction, etc. were collected in accordance with the requirements of the instructions, and human venous blood was collected with EDTA anticoagulant tubes. According to the instructions, the collected blood should not be stored at 2-8°C for more than one week, and at -20°C for no more than one month, and can be transported in a curling box with ice or a foam box with ice. It is recommended to use fresh blood as much as possible. Genomic DNA extraction. Since this kit does not provide human genomic DNA extraction reagents, commercial nucleic acid extraction kits (such as QIAGEN’s DNeasy Blood and Tissue kit) were used to extract human genomic DNA from 200ul whole blood of each patient, and the DNA was extracted using NanoDrop 2000 (Thermo Company) quantified and standardized to 30ng / ul (A1-A10 respec...

Embodiment 3

[0136] Embodiment three: Biological experiment.

[0137] Using an ABI 9700 PCR instrument, according to the instruction manual, 7 polymorphic sites related to the dosage of warfarin were tested.

[0138] The components used in the kit for PCR, PCR product purification and single base extension are:

[0139] serial number

component name

main ingredient

Specification

1

PCR Primer Mix

PCR primers

24ul / tube x1 tube

2

PCR reaction solution

Taq enzyme, dNTP

72ul / tube x1 tube

3

Enzyme digestion reaction solution

SAP enzyme

48ul / tube x1 tube

4

Extension Primer Mix

extension primer

24ul / tube x1 tube

5

Extension reaction solution

Single base elongase, ddNTP

24ul / tube x1 tube

6

positive control

Human Genomic DNA (30ng / ul)

10ul / tube x1 tube

[0140] According to the manual, the specific operation method is as follows:

[0141] 1. PCR amplificatio...

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Abstract

The invention discloses a primer system for detecting 7 gene polymorphic sites related to warfarin dosage. A product prepared based on the primer system can be used for realizing the detection of 7 gene polymorphic sites related to warfarin dosage. The product is used so as to detect the genetypes of a warfarin user at the 7 gene polymorphic sites; and the detection result is combined with other clinical indexes so that a reference can be provided for a clinical doctor to reasonably define the warfarin dosage, and the side effect of a medicament is avoided. According to the invention, the primer system can be used for simultaneously detecting the 7 gene polymorphic sites on different genes in one reaction system; and compared with technologies such as sequence measurement and real-time fluorescencent quantitative PCR (polymerase chain reaction), the primer system disclosed by the invention has the advantages of lower cost, simplicity and convenience in operation and improved accuracy and sensitivity.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a detection method and product for determining a gene polymorphic site (SNP) related to the dose of warfarin, specifically by using multiplex PCR technology, single base extension technology and mass spectrometry Technology, a method for detecting 7 gene polymorphic sites related to warfarin dosage and corresponding kits. Background technique [0002] Warfarin is currently the most widely used oral anticoagulant of dicoumarin derivatives. It is mainly used clinically for the prevention and treatment of thromboembolic diseases, and it also needs to be taken after surgical operations such as heart valve replacement and stent placement. Warfarin to reduce the incidence of venous thrombosis. However, the effective therapeutic range of warfarin is narrow and there are large differences in the dosage among different individuals. To achieve the same effect, the difference between high and lo...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 马庆伟赵洪斌张海燕李京励
Owner 毅新兴业(北京)科技有限公司
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