Method for quickly detecting CYP2C19 genetic polymorphism and kit

A CYP2C19, gene polymorphism technology, applied in the field of molecular biology, can solve the problems of amplification (efficiency, specificity, selectivity, high detection cost, erroneous conclusions, etc., to save DNA extraction steps, high detection Specificity, fast detection effect

Inactive Publication Date: 2017-09-29
BEIJING TIANTAN HOSPITAL AFFILIATED TO CAPITAL MEDICAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The main disadvantages of the sequencing method are: (1) Before the PCR reaction, it is necessary to extract DNA from the sample, check the quality and concentration, the operation is complicated, and there is a risk of confusing the sample
(2) When detecting multiple sites, one sample needs to perform multiple reactions, the detection cost is high, the demand for the amount of template is high, the operation intensity is high, and it is easy to cause operation errors and pollution
The main limiting factors of this method are: a) if the mutated site is a weakly mismatched base sequence, the ARMS primers cannot effectively distinguish the wild-type varian

Method used

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  • Method for quickly detecting CYP2C19 genetic polymorphism and kit
  • Method for quickly detecting CYP2C19 genetic polymorphism and kit
  • Method for quickly detecting CYP2C19 genetic polymorphism and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Embodiment 1: the processing of blood sample

[0055] In this example, 5 sets of blood were subjected to rapid processing after 40-fold dilution. The treatment solution is 20mM Tris-HCl pH8.0, 2mM EDTA, 2mg / ml proteinase K, mix 2ul of blood with 78ul of the treatment solution, mix thoroughly, and then keep warm at 56°C for 5min and 98°C for 2min, the crude sample is Processing complete.

Embodiment 2

[0056] Embodiment 2: the processing of saliva test sample

[0057] In this embodiment, the mucosal cells in the oral cavity were scraped with a special saliva test piece, part of the test piece was cut with scissors or the whole test piece was soaked, wherein the treatment solution was 20mM Tris-HCl pH8.0, 2mM EDTA, 2mg / ml proteinase K, immerse in the required volume, it is advisable to cover all the samples with the treatment solution, and shake vigorously for 1min, the crude sample is processed.

Embodiment 3

[0058] Embodiment 3: Rapid detection of blood samples

[0059] In this example, the blood is rapidly processed and the amplification of the internal reference gene is detected by using the method in Example 1 of the present invention.

[0060] The internal reference sequence is:

[0061] 5'-CGGACTGAAGGAGCTGCCCATGAGAAATTTACAGGGTGAGAGGCTGGGATGCCAAGGCTGGGGGTTCATAAATGCAGACAGCAGTTCCGATGGC-3'

[0062] Forward primer: 5'-CGGACTGAAGGAGCTGCC-3'

[0063] Reverse primer: 5'-GCCATCGGAACTGCTGTCTG-3'

[0064] Detection probe: 5'-Cy5-CCCCAGCCTTGGCATCCCA-BHQ2-3'

[0065] The PCR reaction system is 25 μl, each reaction system contains 2% glycerol, 200 μM each of dATP, dCTP, dGTP, and dTTP, 200 nM each of forward primer and reverse primer, 200 nM detection probe and 1 unit of hot-start Taq DNA polymerization enzyme. One unit refers to the amount of enzyme required to incorporate 10nmoldNTPs at 72°C for 30 minutes.

[0066] ABI 7500Fast fluorescent quantitative PCR instrument was used for ...

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Abstract

The invention relates to the field of molecular biology, discloses a method for quickly detecting CYP2C19 genetic polymorphism and a kit, and provides the method for quickly detecting the CYP2C19 genetic polymorphism and the kit. A sample to be tested is mixed with sample treating fluid, a DNA extraction step is not needed, and simple and easy treatment on samples can be realized in short time; moreover, further through a selective amplification primer and a selective detection probe, effective distinguishing between wild type variants and mutant type variants of the CYP2C19 gene can be realized, and a whole process from treatment of the samples to acquisition of a final detection result is completed within 1 hour; moreover, the accuracy, the specificity and the sensitivity of the detection can be ensured.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a method and kit for rapidly detecting CYP2C19 gene polymorphism. Background technique [0002] Cytochrome P450 (CYP or P450) is one of the main metabolic enzymes in the liver. CYP2C19 is an important member of the second family of P450 enzymes, involved in about 12% of the P450 enzymes responsible for metabolizing drugs, and is an important drug-metabolizing enzyme in the human body. At present, many single nucleotide polymorphisms of CYP2C19 have been found, causing the occurrence of CYP2C19 alleles, among which CYP2C19*2 (G681A, rs4244285), CYP2C19*3 (G636A, rs4986893) and CYP2C19*17 are common in Asian populations allelic type. CYP2C19*2 and CYP2C19*3 can cause the loss of the enzyme activity encoded by the CYP2C19 gene, weaken the ability to metabolize the substrate, and increase the blood drug concentration, thereby causing adverse drug reactions related to the ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6858C12Q2563/107C12Q2527/125C12Q2545/101
Inventor 王拥军王伊龙李伟毕万里王大永崔恒进
Owner BEIJING TIANTAN HOSPITAL AFFILIATED TO CAPITAL MEDICAL UNIV
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