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Kit and method for detecting CYP3A5 gene polymorphism

A gene polymorphism and polymorphism technology, applied in the field of fluorescence quantitative PCR, can solve the problems of low polymorphism sensitivity and specificity, low sensitivity and specificity, time-consuming and labor-intensive, and avoid false positives. Or false negative, good specificity, high accuracy effect

Active Publication Date: 2013-12-11
刘辉
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In view of the problems in the prior art that the sensitivity and specificity of detecting CYP3A5 gene CYP3A5*3, CYP3A5*4, CYP3A5*6 and CYP3A5*7 polymorphisms are not high, and it is time-consuming and labor-intensive, the purpose of the present invention is to provide a A kit for detecting CYP3A5 gene polymorphism
[0007] In order to solve the problems of low sensitivity and specificity and time-consuming and labor-consuming detection of CYP3A5 gene CYP3A5*3 polymorphism, CYP3A5*4 polymorphism, CYP3A5*6 and CYP3A5*7 polymorphism in the existing technology , the present invention has adopted following technical scheme:

Method used

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  • Kit and method for detecting CYP3A5 gene polymorphism
  • Kit and method for detecting CYP3A5 gene polymorphism
  • Kit and method for detecting CYP3A5 gene polymorphism

Examples

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Embodiment 1

[0055] Example 1. Preparation of the kit

[0056] 1. Design of internal reference gene GAPDH, primers and fluorescent probes for detection

[0057] The primers and fluorescent probes specific to the above-mentioned gene sequences were designed according to the gene sequences. The GAPDH gene sequence and the CYP3A5 gene sequence were derived from the National Center for Biotechnology Information Nucleic Acid Database (NCBI), and the GAPDH gene ID was 2597, respectively, and the reference sequence The number is NG_007073.2, and you can also refer to SEQ ID NO: 16 in the sequence listing of the present invention; the CYP3A5 gene ID is 1577, and the reference sequence number is NC_000007.13. Primer 5.0 primer design software was used to design the following primers: specific upstream and downstream primers for CYP3A5 gene CYP3A5*3 polymorphism and specific Taqman fluorescent probe for CYP3A5 gene CYP3A5*3 polymorphism, CYP3A5 gene CYP3A5*4 The specific upstream and downstream pri...

Embodiment 2

[0086] Example 2. Detection of CYP3A5 gene polymorphism with the kit of Example 1

[0087] Take the results of random detection of peripheral blood samples from 30 subjects as an example.

[0088] The detection process for detecting CYP3A5*3 gene polymorphism, CYP3A5*4 gene polymorphism, CYP3A5*6 and CYP3A5*7 gene polymorphism of a subject with the kit of the present invention is as follows: first obtain the Genomic DNA was quickly extracted from the peripheral blood samples of patients; secondly, the fluorescent quantitative PCR reaction solution of the internal reference gene GAPDH and the internal positive control sequence was first prepared, and the internal positive control sequence standard and the internal reference gene GAPDH standard were diluted to copy numbers / mL respectively. 1.0x10 3 , 1.0x10 4 , 1.0x10 5 and 1.0x10 6 , make the standard curve of the internal positive control sequence standard and the standard curve of the internal reference gene GAPDH standar...

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Abstract

The invention relates to a kit and a method for detecting CYP3A5 gene polymorphism, belonging to the field of fluorescent quantitative PCR (Polymerase Chain Reaction). The kit comprises primers for detection and fluorescent probes for detection, wherein the primers for detection and the fluorescent probes for detection comprise at least one group of specific upstream and downstream primers for detecting CYP3A5*3 polymorphism of the CYP3A5 gene as well as a Taqman fluorescent probe, specific upstream and downstream primers for detecting CYP2C19*4 polymorphism of the CYP3A5 gene as well as a Taqman fluorescent probe, specific upstream and downstream primers for detecting CYP2C19*6 polymorphism of the CYP3A5 gene as well as a Taqman fluorescent probe, and specific upstream and downstream primers for detecting CYP3A5*7 polymorphism of the CYP3A5 gene as well as a Taqman fluorescent probe. The detection sensitivity and specificity of the kit are remarkably improved, the detection time is short, and prediction of drug dose and curative effect is facilitated.

Description

technical field [0001] The invention relates to the field of fluorescence quantitative PCR, in particular to a kit and method for detecting CYP3A5 gene polymorphism. Background technique [0002] The CYP3A5 gene is located on human chromosome 7, with a total length of 31.8kb and 13 exons, encoding 502 amino acids. It is the main extrahepatic expression form of CYP3A subfamily. It shows higher expression levels than CYP3A4 gene in lung, kidney, breast, prostate and polymorphonuclear leukocytes, but has a similar coding amino acid sequence to CYP3A4 gene. substrates and the same regulatory pathways. CYP3A5 wild-type is defined as CYP3A5*1. Only individuals who carry at least one CYP3A5*1 allele can express CYP3A5 protein normally. For details of CYP3A5 gene polymorphism, please refer to the CYP allele database (http: / / www.cypalleles.ki .se). The single nucleotide polymorphisms (single nucleotide polymorphisms, SNPs) of CYP3A5 were first discovered in 1996 by Jounaidi et al....

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 刘辉
Owner 刘辉
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