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CYP2C19 gene detection kit, amplification method and detection method

A CYP2C19, gene detection technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve problems such as liver damage, and achieve the effect of reducing operation steps, avoiding false negative results, and avoiding human error.

Inactive Publication Date: 2013-07-03
UNION STEMCELL & GENE ENG +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Sodium valproate and magnesium valproate are currently the first choice drugs for the treatment of generalized or partial epilepsy, but the metabolites of valproic acid have certain hepatotoxicity and damage the liver

Method used

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  • CYP2C19 gene detection kit, amplification method and detection method
  • CYP2C19 gene detection kit, amplification method and detection method
  • CYP2C19 gene detection kit, amplification method and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Step 1: Preparation of whole blood cell lysate

[0056] Take 300 μl of peripheral venous blood from the subject, add 700 μl of cell lysate, invert and mix 5 times, centrifuge at 12,000 rpm for 1 minute, discard the supernatant, and place the centrifuge tube upside down on clean absorbent paper for 2 minutes to ensure that the precipitate remains in the tube , add 300 μl double distilled water, vortex and shake to form a cell lysate suspension.

[0057] Step 2: PCR amplification reaction

[0058] 1. Configure the wild-type reaction system: add 4.8 μl of cell lysis suspension, 5 μl of 2× wild-type amplification buffer, and 0.2 μl of polymerase (2.5 U / μl) into the PCR tube to form an independent reaction system. Mix well after adding the sample. Evenly, briefly centrifuge, see the table below for details:

[0059] 2× wild-type amplification buffer solution

5μl

Cell Lysis Suspension

4.8μl

Polymerase (2.5U / μl)

0.2μl

[0060] 2. Co...

Embodiment 2

[0073] Step 1: Extraction and dilution of whole blood genomic DNA

[0074] Take 300 μl of peripheral venous blood from the subject, and extract whole blood genomic DNA according to the instructions of the whole blood genomic DNA extraction kit. Measure the concentration of DNA with a spectrophotometer and dilute to 15-20 ng / μl.

[0075] Step 2: PCR amplification reaction

[0076] 1. Configure the wild-type reaction system: add 4.8 μl of cell lysis suspension, 5 μl of 2× wild-type amplification buffer, and 0.2 μl of polymerase (2.5 U / μl) into the PCR tube to form an independent reaction system. Mix well after adding the sample. Evenly, briefly centrifuge, see the table below for details:

[0077] 2× wild-type amplification buffer solution

5μl

Cell Lysis Suspension

4.8μl

Polymerase (2.5U / μl)

0.2μl

[0078] 2. Configure the mutant reaction system: add 4.8 μl of cell lysis suspension, 5 μl of 2× mutant amplification buffer and 0.2 polym...

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Abstract

The present invention discloses a gene detection kit using the multiplex PCR technology combined with the SNP sensitive molecular switch technology for genotyping of cytochrome 4502C19 gene, an amplification method and a detection method. Three common polymorphic sites on the CYP2C19 gene significantly changing the product activity are genotyped by the kit, wherein the three common polymorphic sites include: rs4244285SNP site, rs4986893 site and rs12248560 site. The kit includes a wild-type 2*PCR buffer, a 2*mutant amplification buffer, and a polymerase. The two buffers respectively includes a sequence-specific primer and an internal reference primer corresponding to SNP wild and mutant phenotype, and can complete genotyping for the three SNP sites in two multiplex PCR reactions, and thus the CYP2C19 gene is genotyped, and molecular biological evidence is provided for the gene-expressed enzyme activity prediction.

Description

technical field [0001] The invention relates to a kit for detecting SNP sites and a PCR amplification method thereof, in particular to a kit for detecting SNP sites related to CYP2C19 enzyme activity using multiplex PCR combined with molecular switch technology, and a multiplex PCR amplification method and detection method thereof . Background technique [0002] Cytochrome P450 is a group of structurally and functionally related isozymes encoded by superfamily genes, which play an important role in drug metabolism. Most drugs require biotransformation after entering the body. Biotransformation mainly includes two processes of phase I reaction and phase II reaction. The metabolic enzymes involved in the phase I reaction are mainly cytochrome P450 enzymes in the endoplasmic reticulum of liver cells. Cytochrome oxidase P450 2C19 (CYP2C19), also known as S-mephenytoin hydroxylase, is a member of cytochrome P450. There are obvious individual differences in its activity, which ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 韩俊领杜宏伟周毓玲崔丽娟
Owner UNION STEMCELL & GENE ENG
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