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Primer, probe and kit for detecting gene polymorphism of CYP2C9 and VKORC1

A gene polymorphism and kit technology, applied in the direction of DNA / RNA fragments, recombinant DNA technology, etc., can solve the problems of complicated PCR-PFLP steps, inability to detect homozygous mutations, easy to contaminate the interpretation of results, etc., to achieve amplification The effect of better effect, fast detection speed and low cost

Inactive Publication Date: 2017-02-15
武汉海吉力生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

DNA sequencing is the gold standard for detection, but it requires high technical requirements, is easy to pollute, and is time-consuming and laborious to interpret the results
PCR-PFLP has complicated steps and low sensitivity
HRM is simple and fast, but cannot detect homozygous mutations, and is prone to false positives and false negatives
The equipment needed for Genechips is expensive, and the cost of chips is high, making it difficult to popularize

Method used

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  • Primer, probe and kit for detecting gene polymorphism of CYP2C9 and VKORC1
  • Primer, probe and kit for detecting gene polymorphism of CYP2C9 and VKORC1
  • Primer, probe and kit for detecting gene polymorphism of CYP2C9 and VKORC1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Embodiment 1 kit composition

[0063] The composition of the detection kit of the present invention is listed in the following two types, see Table 1 and Table 2 respectively.

[0064] Table 1 Kit composition one

[0065]

[0066]

[0067] Table 2 Kit composition two

[0068]

[0069] Fast masterPremix is ​​a product of ABI (ie, AppliedBiosystems, an American Applied Biosystems company), which integrates NDA polymerase and PCR buffer. It is beneficial to advance the Ct value during fluorescent quantitative PCR, and the detection result is easier to judge.

Embodiment 2

[0070] Embodiment 2: Extraction of clinical sample DNA

[0071] 1. EDTA anticoagulant

[0072] In this part of the experiment, genomic DNA was extracted from EDTA anticoagulated blood and quantified as a template for PCR detection. The Genomic Blood DNA Extraction Kit from Tiangen Company was used, and the details are as follows.

[0073] 1. Processing blood material:

[0074] a. When the volume of the blood sample is less than 200 μL, add buffer GS to make up the volume to 200 μL, and then proceed to the next step of the experiment (if the volume of the blood sample is 200 μL, the next step of the experiment can be carried out directly without adding GS).

[0075] b. When the volume of the blood sample exceeds 200 μL, it needs to be treated with cell lysate CL. The specific steps are as follows: add 1 to 2.5 times the volume of cell lysate CL to the sample, mix it upside down, and centrifuge at 10,000 rpm (~11,500×g) After 1 min, remove the supernatant and leave the cell n...

Embodiment 3

[0102] Embodiment 3: Real-time fluorescence PCR method amplifies clinical sample DNA

[0103] This example uses the primers and probes provided by the present invention to amplify the DNA sample extracted in Example 2.

[0104] Fluorescent quantitative PCR reaction system is:

[0105] PCR reaction solution:

[0106] MgCl 2 : 2.0~5.0mmol,

[0107] dNTP: 0.2~0.8mmol,

[0108] Each primer: 0.1~1.0μmol,

[0109] Each probe: 0.1~1.0μmol,

[0110] Fast master premix: 6-10 μL or hot start enzyme 0.3-0.6 μL,

[0111] Template: 10 μL,

[0112] Total volume: 40 μL.

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Abstract

The invention discloses a primer, a probe and a kit for detecting gene polymorphism of CYP2C9 and VKORC1. The primer comprises a gene detection specificity primer sequence of three loci YP2C9*2, CYP2C9*3 and VKORC1 and a probe sequence (SEQ ID NO.1-12). An ARMS primer is used to differentiate field and mutant genes, so that the primer has the advantages of high sensitivity and high specificity and is suitable for various sample types and high in detection result accuracy. In addition, the ARMS primer is quick and simple to synthesize, low in synthesis cost and better in amplification effect, and production cost can be lowered effectively. The kit is high in detection speed and quite convenient, and the whole detection process only takes 90 minutes. The whole kit does not contain toxic and harmful substances, thereby being harmless to operating personnel and environment and suitable for wide application in clinic detection and the like.

Description

technical field [0001] The invention relates to the technical field of gene polymorphism detection, in particular to primers, probes and kits for detecting CYP2C9 and VKORC1 gene polymorphisms. Background technique [0002] Warfarin is currently a coumarin-based oral anticoagulant widely used clinically. It has been used in the anticoagulant therapy of many diseases, such as the primary and secondary prevention of venous thromboembolic disease (VTE), atrial fibrillation, etc. Prevention of thromboembolism, valvular disease, artificial valve replacement, and intracardiac thrombosis, etc., but the clinical efficacy and adverse reactions vary greatly from individual to individual, and the dose is difficult to control, especially in the initial stage of warfarin anticoagulant therapy, which can easily lead to severe bleeding complications. It is estimated that among patients taking warfarin, 15.2% of them experience bleeding side effects every year, of which fatal bleeding acco...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 史学晖胡利红段卫涛赵平锋
Owner 武汉海吉力生物科技有限公司
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