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Detection kit for CYP2C19 and ABCB1 genes

A gene and reagent technology, applied in recombinant DNA technology, microbial assay/inspection, biochemical equipment and methods, etc., can solve problems such as high cost and complicated operation

Active Publication Date: 2017-08-11
BIOYONG TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the above sites do not include ABCB1 (3435C>T), and all CYP2C19 sites related to clopidogrel have not been studied, and at the same time, in the process of nucleic acid mass spectrometry detection, the interference of the multiple amplification process has great influence on the final obtained The extension product is also affected, so the inventors tried to find a new combination of CYP2C19 site and ABCB1 site, and redesigned a new primer system
[0014] In summary, the current technical problems are: lack of methods and products that can effectively detect multiple gene polymorphisms related to clopidogrel resistance at one time, common detection technologies, such as sequencing, real-time fluorescent quantitative PCR, etc. It is necessary to detect the sites one by one. When there are many sites, the operation is complicated and the cost is high; another example is that the known nucleic acid mass spectrometry detection technology fails to disclose the above combined sites and specific primer systems. At the same time, because the same nucleic acid mass spectrometry During the detection process, the interference of multiplex PCR on the extension product

Method used

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  • Detection kit for CYP2C19 and ABCB1 genes
  • Detection kit for CYP2C19 and ABCB1 genes
  • Detection kit for CYP2C19 and ABCB1 genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0104] Example 1: Primer design and synthesis.

[0105]For CYP2C19 genes rs4244285(CYP2C19*2 681G>A), rs4986893(CYP2C19*3 636G>A), rs28399504(CYP2C19*4 1A>G), rs56337013(CYP2C19*5 1297C>T), rs12926YP27 >T) and ABCB1 gene rs1045642 (3435C>T) and other 6 polymorphic sites of genes related to drug metabolism were designed corresponding to specific PCR primer core sequences (SEQ ID No: 1 to SEQ ID No: 12) and specific extension primers Core sequence (SEQ ID No: 13 to SEQ ID No: 18).

[0106] Among them, in order to prevent the PCR primers from entering the detection window of the mass spectrometer and interfering with the detection effect, a certain number of bases can be added to the core sequence (SEQ ID No: 1 to SEQ ID No: 12) at the 5' end of each PCR primer , common such as 10bp tag (ACGTTGGATG), to increase the molecular weight of the PCR primer, thereby exceeding the detection window of the mass spectrometer.

[0107] The relevant primers were synthesized in Shanghai Jier...

Embodiment 2

[0108] Embodiment 2: sample DNA extraction.

[0109] A total of 10 clinical patients 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 stored at ‐20°C for no more than one month, and can be transported in a curler 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, a commercial nucleic acid extraction kit (such as QIAGEN’s DNeasy Blood and Tissue kit) was used to extract human genomic DNA from 200 μl whole blood of each patient, and the DNA was extracted using NanoDrop 2000 (Thermo Company) quantified, and standardized to 30ng / μl (respectively C1‐C10). Among them, the kit is recom...

Embodiment 3

[0110] Embodiment three: Biological experiment.

[0111] Using ABI 9700 PCR instrument, according to the instructions, the 6 genes related to clopidogrel resistance gene and the polymorphic site of ABCB1 gene were tested.

[0112] The components used in the kit for PCR, PCR product purification and single base extension are shown in Table 3:

[0113] Table 3. Kit composition

[0114] serial number component name main ingredient Packing Specifications 1 Reaction solution I dNTPs, Tris, MgCl 2

270μL / tube×1 tube 2 Enzyme I PCR enzyme, UNG enzyme 22μL / tube×1 tube 3 Amplification primer PCR primers 100μL / tube×1 tube 4 Reaction solution II Tris, MgCl 2

170μL / tube×1 tube 5 Enzyme II SAP enzyme 30μL / tube×1 tube 6 Reaction solution III ddNTPs, Tris, MgCl 2

100μL / tube×1 tube 7 Enzyme III elongase 4μL / tube×1 tube 8 extension primer extension primer 100μL / tube×1 tube 9 positiv...

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Abstract

The invention provides a primer combination for detecting gene polymorphic sites relevant to clopidogrel resistance, wherein the sites comprise the polymorphic sites including CYP2C19*2, *3, *4, *5, *17 and the polymorphic site 3435C>T of ABCB1. According to the scheme, multiple PCR primers are utilized for amplifying gene segments where relevant gene loci are located, after the amplification product is treated, single base extension is carried out on the to-be-detected loci, then molecular weight difference detection is carried out on the extension product by adopting a flight time mass spectrum, through data analysis, the result of the relevant gene for the clopidogrel resistance of a patient is rapidly detected. The invention further provides a detection product prepared by adopting the primer combination and applications of the detection product, and thus references are provided for the individualized medication of clopidogrel.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a technology for detecting the SNP site of a gene related to clopidogrel self-administration, specifically using multiplex PCR technology, single base extension technology and mass spectrometry technology to detect clopidogrel-related genes. Products and uses for detection of 6 genetic polymorphic sites. Background technique [0002] Clopidogrel (Clopidogrel) is a new antiplatelet drug of the thienopyridine class, which can be used in combination with aspirin for the prevention and treatment of recurrent acute coronary syndrome (ACS) ischemic events, implantation of metal stents or drug-releasing stents Standard therapy for patients undergoing percutaneous coronary intervention (PCI) and acute myocardial infarction. Clopidogrel is a drug precursor (prodrug), which itself is inactive. About 50% of clopidogrel is absorbed through the gastrointestinal tract and is rapidly metabolized in ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q1/6883C12Q2600/106C12Q2600/156C12Q2600/16C12Q2531/113C12Q2537/143C12Q2533/101C12Q2565/627
Inventor 马庆伟张海燕钟逾杨宇凤王宇涵吴娜拉胡李清林燕
Owner BIOYONG TECH
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