Composition for detecting CYP2C19 gene polymorphism and its application

A technology of CYP2C19 and gene polymorphism, applied in the direction of recombinant DNA technology, microbial measurement/testing, biochemical equipment and methods, etc., can solve the problems of adding probes, low accuracy and specificity, and avoid cross-contamination , accurate results, and improved simplicity

Inactive Publication Date: 2017-10-24
北京鑫诺美迪基因检测技术有限公司
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

And on this basis, the probe is modified by locking nucleic acid and secondary structure, increasing the Tm value of the probe, and further improving the specificity of the probe binding. Finally, through the adjustment of the above system, the accuracy and specificity are evenly balanced. It can reach 100%, and the minimum detection concentration can reach 1ng / μL, which solves the problem of low accuracy and specificity in the detection of CYP2C19 gene polymorphism in the past

Method used

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  • Composition for detecting CYP2C19 gene polymorphism and its application
  • Composition for detecting CYP2C19 gene polymorphism and its application
  • Composition for detecting CYP2C19 gene polymorphism and its application

Examples

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Embodiment 1

[0078] Embodiment 1: On the basis of multiplex fluorescent PCR, the present invention is formulated into a premixed primer composition for detection sites. The extracted sample can be tested directly, which makes the detection step easier.

[0079] Specific steps are as follows:

[0080] 1) Genomic DNA was extracted from the whole blood sample (using a commercial peripheral blood genomic DNA extraction kit).

[0081] 2) Measure the concentration of the extracted genomic DNA, and dilute the concentration to 10-20 ng / μL for subsequent PCR reaction process.

[0082] 3) Composition of the reaction system: dNTPs, Mg 2+ , DNA polymerase buffer, reverse transcription buffer, gene primer pair, DNA polymerase, etc.

[0083] 4) PCR reaction conditions: UDG enzyme reaction at 37°C for 2min; pre-denaturation at 95°C for 3min; denaturation at 94°C for 15s, annealing and extension at 60°C for 35s, 40 cycles. The addition of UDG enzyme prevents the contamination of uracil in the system. ...

specific Embodiment approach 2

[0108] Collect 200 peripheral blood control samples from Wuhan Tongji Hospital, and use the above-mentioned finished kit for detection. The specific operation steps are as described in Example 1.

[0109]

[0110]

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Abstract

The invention discloses a composition for detecting CYP2C19 gene polymorphism and its application. The single nucleotide polymorphism site in the CYP2C19 gene is detected through FAM, HEX, ROX (modified) three-channel multiple PCR reaction. In order to improve the detection simplicity and specificity, one-tube parting detection is realized through the technical manner of a probe parting, thus the whole operation and the reaction process are simplified; on this basis, locked nucleic acid modification and second-grade structure modification are performed on the probe to increase the Tm value of the probe, and further improve the probe combining specificity; finally, the lowest detection limit, accuracy and the specificity of the whole kit are improved through adjusting the mixture ratio of the primer pair, dosage of Taq enzyme, dosage of magnesium ion and others; the lowest detection concentration of the kit can reach 1 mg/mu l; the accuracy and the specificity can reach 100%.

Description

technical field [0001] The invention belongs to the technical field of gene detection, and in particular relates to a technology for detecting CYP2C19 gene polymorphism. Background technique [0002] Clopidogrel, a thienopyridine antiplatelet drug, is a second-generation ADP receptor antagonist. Clopidogrel is an inactive prodrug that needs to be activated by the cytochrome P450 enzyme system in liver cells, about 85% of which are esterified into inactive metabolites and metabolized by the intestinal tract, and only about 15% of clopidogrel is Activated to generate active metabolites to exert their antiplatelet pharmacological effects. [0003] Clopidogrel is the basic antithrombotic drug for the treatment of acute coronary syndrome and percutaneous coronary intervention, but 4% to 30% of patients experience a decline in the efficacy of clopidogrel during treatment, and even clopidogrel resistance. Clopidogrel is a prodrug, which mainly depends on the metabolism of CYP2C19...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q1/6883C12Q2600/106C12Q2600/156C12Q2600/16C12Q2531/113C12Q2537/143C12Q2563/107
Inventor 刘沛王林海
Owner 北京鑫诺美迪基因检测技术有限公司
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