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Composition for detecting CYP2C19 and VKORC1 gene polymorphism and its application

A gene polymorphism and composition technology, applied in the field of detecting CYP2C9 and VKORC1 gene polymorphism, can solve the problems of low accuracy and specificity, increase probes, etc., achieve accurate results, avoid cross-contamination, and accurately and effectively detect Effect

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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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 CYP2C9 and VKORC1 gene polymorphisms in the past

Method used

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  • Composition for detecting CYP2C19 and VKORC1 gene polymorphism and its application
  • Composition for detecting CYP2C19 and VKORC1 gene polymorphism and its application
  • Composition for detecting CYP2C19 and VKORC1 gene polymorphism and its application

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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.

[0084] 5) Preparation of PCR reaction system:

[0085] PCR amplification MIX...

specific Embodiment approach 2

[0107] 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.

[0108]

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Abstract

The invention discloses a composition for detecting CYP2C19 and VKORC1 gene polymorphism and its application. The single nucleotide polymorphism site in the CYP2C19 and VKORC1 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 CYP2C9 and VKORC1 gene polymorphisms. Background technique [0002] Warfarin is an indirect-acting coumarin-based oral anticoagulant that exerts its anticoagulant effect by inhibiting the synthesis of several coagulation factors by vitamin K in liver cells. Warfarin can change the viscous state of blood and is clinically used to prevent and treat thrombosis. The normal dose of warfarin can anticoagulate, if the dose is insufficient, it cannot achieve the therapeutic effect of preventing blood clots; if the dose is too high, it will easily lead to various bleeding. Once bleeding occurs in the patient, the dose of warfarin must be reduced or discontinued, and the subsequent consequence is that the anticoagulant effect is weakened or stopped, causing the patient to form a thrombus again. Due to differences in drug metabolism among pati...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/6883C12Q2600/106C12Q2600/156C12Q2563/107C12Q2537/143
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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