Kit for quickly detecting polymorphism of human CYP2C19 gene and using method of kit

A CYP2C19 and gene polymorphism technology, applied in the field of biomedicine, can solve the problems of high price, easy false positive, and difficult primer design, achieve low false positive and false negative rates, reduce the risk of false positives, and achieve high The effect of flux detection

Active Publication Date: 2014-12-17
WUHAN HEALTHCHART BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Advantages: low cost, intuitive results; Disadvantages: Some polymorphic sites do not have suitable restriction enzyme sites, and mutations need to be introduced manually. In addition, it is easy to cause product contamination, resulting in false positive results. At the same time, the operation is cumbersome, Long test cycle
Disadvantages: PCR products need to be analyzed later, and the operation is cumbersome; in addition, the results are not easy to interpret, and false positives are prone to occur
Disadvantages: It is difficult to design primers, and when there are a large number of GC or AT seq...

Method used

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  • Kit for quickly detecting polymorphism of human CYP2C19 gene and using method of kit
  • Kit for quickly detecting polymorphism of human CYP2C19 gene and using method of kit
  • Kit for quickly detecting polymorphism of human CYP2C19 gene and using method of kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] Example 1 Probe, Primer and PNA Synthesis

[0091] The probes, primers and PNA of the present invention were all synthesized in Shanghai Sangong Engineering Co., Ltd.

Embodiment 2

[0092] Embodiment 2 kit composition

[0093] The configuration of the kit is configured in the form of PCR8 strip tubes, and only the sample / control substance and ddH need to be added during use. 2 O can complete the experimental configuration, this configuration is more convenient and quicker, and is the preferred configuration.

[0094] In this embodiment, the kit is configured in the form of PCR8 tube strips, and the configuration of the kit and the composition of the PCR8 tube strips are shown in Table 1 and Table 2 below.

[0095] Table 1 Kit composition

[0096]

[0097] Table 2 Composition of PCR8 strips

[0098]

[0099]

[0100] The above-mentioned PCR8 tubes A and B contain PCR reaction solutions related to the detection of human CYP2C19 gene 681 typing, PCR8 tubes C and D contain PCR reaction solutions related to the detection of human CYP2C19 gene 836 typing, PCR8 tubes E and F contains the PCR reaction solution related to the detection of human CYP2C19...

Embodiment 3

[0101] Embodiment 3 uses the operating steps of kit

[0102] 1. Human peripheral blood total DNA extraction, the extraction kit uses the whole blood DNA extraction kit of Tiangen Biochemical Biotechnology Company, the specific operation steps are carried out in strict accordance with the kit instructions, and the human total DNA template (that is, the test sample) is obtained after the extraction is completed;

[0103] 2. Take out one PCR8 tube in the kit, add 2 μL of the test sample to tubes A to F, take another PCR8 tube, add 2 μL of the corresponding positive control substance to tubes A to F, and then Take one PCR8 strip tube, add 2 μL of negative control to tubes A~F, and finally add ddH to tubes A~F of all PCR8 strip tubes 2 O to make up 20 μL.

[0104] 3. Put the PCR8 strip tube into the fluorescent PCR instrument, and perform fluorescent PCR amplification detection; the reaction conditions are: denaturation at 94°C for 3 minutes; denaturation at 94°C for 30 seconds; a...

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Abstract

The invention belongs to the gene detecting technology in clinical detecting technologies of the bio-medical field and particularly relates to a kit for quickly detecting polymorphism of a human CYP2C19 gene and a using method of the kit. The kit comprises three pairs of primers, three probes, six PNA (pentose nucleic acid) sequences, a pair of beta-Actin interior label primers and an interior label probe, and further comprises a PCR (polymerase chain reaction) buffer solution of Mg<2+>, a dNTP mixture, Taq enzyme and ddH2O. The kit can be utilized to carry out qualitative detection on a polymorphic site of the CYP2C19 gene and carry out two-tube PNA-PCR fluorescent amplified reaction according to the SNP site and can carry out result judgment according to a condition whether a two-tube fluorescence curve is started or not without producing personal errors, so that a false positive rate and a false negative rate are low. And moreover, the kit can be used to realize high-flux detection easily and is capable of clinical use.

Description

technical field [0001] The invention belongs to the gene detection technology in the clinical detection technology in the field of biomedicine, and specifically relates to a kit for rapidly detecting human CYP2C19 gene polymorphism and a use method thereof. Background technique [0002] Cytochrome P450 (Cytochrome P450, CYP450) is an isoenzyme encoded by a group of structurally and functionally related superfamily genes (Superfamily gene). For humans, it mainly exists in liver cell microsomes. In the CYP450 superfamily, CYP2 is the largest family, with 15 subfamilies (CYP2A to 2Q), which metabolize about 20% of the drugs currently used clinically, among which CYP2A, CYP2C, CYP2D and CYP2E all have genetic polymorphisms and races difference. CYP2C accounts for about 20% in human liver, and CYP2C8, 2C9 / 10, 2C18 and 2C19 have been cloned. In recent years, the research on the correlation between the genotype of CYP450 and the activity of drug-metabolizing enzymes in different ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6858C12Q2531/113C12Q2537/163C12Q2561/101
Inventor 叶伦王宁刘金水李雪梅李倩陈刚
Owner WUHAN HEALTHCHART BIOLOGICAL TECH
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