Probes and kit for detecting human CYP2C9 (Cytochrome P450 2C9) and VKORC1 (Vitamin K epoxide reductase complex subunit 1) gene polymorphism

A technology for gene polymorphism and detection probes, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of complicated operation, long detection process, high cost, and achieve linear detection Wide, high accuracy, the effect of eliminating pollution

Inactive Publication Date: 2015-03-11
HELIXGEN GUANGZHOU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method can carry out high-density gene detection, but it is expensive, cumbersome to ope

Method used

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  • Probes and kit for detecting human CYP2C9 (Cytochrome P450 2C9) and VKORC1 (Vitamin K epoxide reductase complex subunit 1) gene polymorphism
  • Probes and kit for detecting human CYP2C9 (Cytochrome P450 2C9) and VKORC1 (Vitamin K epoxide reductase complex subunit 1) gene polymorphism
  • Probes and kit for detecting human CYP2C9 (Cytochrome P450 2C9) and VKORC1 (Vitamin K epoxide reductase complex subunit 1) gene polymorphism

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 CYP2C9 gene *2 (C430T) site, *3 (A1075C) site and VKORC1 gene (G-1639A) detection kit

[0030] This example designs specific primers and probes for CYP2C9 gene *2 (C430T) site, *3 (A1075C) site and VKORC1 gene (G-1639A), and reagents containing specific primers and specific probes box. The specific composition is as follows.

[0031] Table 1 Primer and probe sequences of human CYP2C9 and VKORC1 gene polymorphism detection kit

[0032]

[0033]

[0034] Using a kit including the above primers and fluorescent probes for simultaneous detection, the method is as follows:

[0035] 1. Sample processing and DNA extraction:

[0036]The test sample is EDTA anticoagulated whole blood sample, and the sample should be stored at room temperature for no more than 1 week, at 4°C for no more than 1 month, and at -20°C for no more than 7 weeks, and the blood sample should be frozen and thawed no more than 5 times. Use Guangzhou Haozhi DNA Extraction Kit (Cat. No.: DT...

Embodiment 2

[0055] In this example, blood clinical samples (concentration range of 2ng / μL-60ng / μL) and CYP2C9*2 heterozygous plasmid (sequence derived from NCBI serial number: NG_008385.1) were used as standard products (plasmid diluted to 20fg / μL) As an example to illustrate the actual clinical sample detection results of the present invention.

[0056] The primers, probes and detection methods for the CYP2C9*2 locus are the same as those described in Example 1, and will not be repeated here.

[0057] The results showed that all types can be effectively interpreted, and the clinical samples were verified to be completely consistent in type by the gold standard Sanger method gene sequencing. The experimental results are attached figure 1 a, 1b, 1c.

Embodiment 3

[0059] In this example, blood clinical samples (concentration range of 2ng / μL-60ng / μL) and CYP2C9*3 heterozygous plasmid (sequence derived from NCBI serial number: NG_008385.1) were used as standard products (plasmid diluted to 20fg / μL) as Example illustrates the actual clinical sample detection results of the present invention.

[0060] The primers, probes and detection methods for the CYP2C9*3 locus are the same as those described in Example 1, and will not be repeated here.

[0061] The results showed that all types can be effectively interpreted, and the clinical samples were verified to be completely consistent in type by the gold standard Sanger method gene sequencing. The experimental results are attached figure 2 a, 2b, 2c.

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Abstract

The invention discloses a kit for detecting human CYP2C9 (Cytochrome P450 2C9) and/or VKORC1 (Vitamin K epoxide reductase complex subunit 1) gene polymorphism. The kit comprises a fluorescence detection probe I represented as SEQ ID NO:3, a fluorescence detection probe II represented as SEQ ID NO:6 and/or a fluorescence detection probe III represented as SEQ ID NO:9, wherein the fluorescence detection probe I comprises an amplimer for C43oT site of a CYP2C9 gene as well as a base; the fluorescence detection probe II comprises an amplimer for A1075C site of the CYP2C9 gene as well as a base; and the fluorescence detection probe III comprises an amplimer for G-1639A site of a VKORC1 gene as well as a base. Specific primers, fluorescence detection probes and an optimized detection system are adopted, the three gene types can be detected separately or synchronously, the detection linearity is wide, a 25 mu L detection system can detect 2ng-600ng human genomic DNA (deoxyribonucleic acid), the accuracy is high, and the detection result is completely consistent with that of the Sanger sequencing method.

Description

technical field [0001] The invention belongs to the field of biological detection, in particular to a probe and a kit for detecting human CYP2C9 and VKORC1 gene polymorphisms. Background technique [0002] Warfarin (Warfarin) belongs to the coumarin class of oral anticoagulant drugs, and is the first-line drug for the treatment of blood embolism diseases (such as heart valve replacement, atrial fibrillation, venous thrombosis and pulmonary infarction, etc.). CYP2C9 is the main metabolizing enzyme of warfarin, and certain genotypes of its SNP will cause the enzyme activity of CYP2C9 to decrease, so that the elimination of warfarin in the body will be slowed down. Warfarin inhibits the synthesis of coagulation factors II, VII, IX, and X involved in vitamin K by inhibiting vitamin epoxide reductase (VKOR), thereby exerting an anticoagulant effect. Therefore, an important subunit gene of VKOR (VKORC1) SNPs can also cause significant differences in individual sensitivity to warf...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6883C12Q2600/106C12Q2600/156
Inventor 曾庆张中满张亚旭张颖芬
Owner HELIXGEN GUANGZHOU
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