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Kit for quickly detecting polymorphism of human VKORC1 and CYP2CP genes and using method of kit

A gene polymorphism and kit technology, applied in the field of biomedicine, can solve problems such as expensive, prone to false positives, and difficult primer design, so as to achieve low false positive and false negative rates, reduce the risk of false positives, and realize The effect of high-throughput detection

Active Publication Date: 2015-01-21
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 sequences at polymorphic sites, it is often helpless, and it is difficult to judge the results
Advantages: One SNP site genotype only needs one tube of PCR to complete the detection; Disadvantages: It is not easy to interpret the difference in Tm value according to the HRM melting curve, and the difference in the peak diagram of the melting curve of different genotypes is not obvious
Disadvantages: Taqman probes need to use MGB-Taqman probes. When the upstream and downstream of the SNP site contain a large number of GC or AT bases, the design and synthesis of MGB-Taqman probes are very difficult and expensive

Method used

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  • Kit for quickly detecting polymorphism of human VKORC1 and CYP2CP genes and using method of kit
  • Kit for quickly detecting polymorphism of human VKORC1 and CYP2CP genes and using method of kit
  • Kit for quickly detecting polymorphism of human VKORC1 and CYP2CP genes and using method of kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] Example 1 Probe, primer and PNA synthesis

[0090] The probes, primers and PNA of the present invention were all synthesized in Shanghai Sangong Engineering Co., Ltd.

Embodiment 2

[0091] Embodiment 2 kit composition

[0092] 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.

[0093] 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.

[0094] Table 1 Kit composition

[0095]

[0096] Table 2 Composition of PCR8 strips

[0097]

[0098]

[0099] The above-mentioned PCR8 tubes A and B contain PCR reaction solutions related to the detection of human VKORC1 gene-1639 typing, PCR8 tubes C and D contain PCR reaction solutions related to the detection of human CYP2C9 gene 430 typing, and PCR8 tube E and F contain the PCR reaction solution related to the detection of human CYP2C9...

Embodiment 3

[0100] Example 3 Operation steps using the kit

[0101] 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;

[0102] 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.

[0103] 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; anneal...

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Abstract

The invention belongs to the gene detecting technology in the clinical detecting technology in the bio-medical field and particularly relates to a kit for quickly detecting polymorphism of human VKORC1 and CYP2CP genes and a using method of the kit. The kit comprises three pairs of primers, three probes, six PNA sequences, 1 pair of beta-Actin interior label primers and one interior label probe, further comprises Mg<2+> PCR (polymerase chain reaction) buffer liquor, a dNTP mixture, Tag enzyme and ddH2O. The kit disclosed by the invention can be used for performing qualitative detection on polymorphic sites of the VKORC1 and CYP2C9 genes, performing two-tube PNA-PCR fluorescent amplification reaction according to an SNP site and performing result judgment according to the condition whether a two-tube fluorescent curve is formed or not without producing manual errors, so that the false positive rate and the false negative rate are low. The kit disclosed by the invention can easily realize high-flux detection and is capable of satisfying clinical use very well.

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 VKORC1 and CYP2C9 gene polymorphisms and a use method thereof. Background technique [0002] Warfarin is currently a widely used coumarin oral anticoagulant for oral anticoagulant therapy in the prevention and treatment of thromboembolic diseases. However, the dosage window of warfarin is narrow, and the required dosage of different patients can vary by more than ten times. Current studies have found that the effects of CYP2C9 and vitamin K epoxide reductase (VKOR) gene polymorphisms on warfarin therapeutic doses are gradually being recognized, among which VKORC1 (-1639G / A), CYP2C9*2 (430C / T) and CYP2C9*3(1075A / C) polymorphisms are most closely related to the dose of warfarin therapy. These polymorphisms are all related to the damage of warfarin metabolizing enzymes, whic...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6858C12Q2525/107C12Q2561/101C12Q2545/113
Inventor 叶伦王宁刘金水李雪梅李倩陈刚
Owner WUHAN HEALTHCHART BIOLOGICAL TECH
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