Fluorescent polymerase chain reaction (PCR) kit for detecting CYP2C19 genotypes

A technology of CYP2C19 and genotyping, which is applied in the field of fluorescent PCR kits and genotypes at two sites, which can solve the problems of high requirements for operators and laboratory conditions, many factors affecting the results, expensive reagents and instruments, etc. , to achieve the effects of avoiding reaction product pollution, short reaction time, and intuitive results

Inactive Publication Date: 2012-07-04
CHANGSHA 3G BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this method has cumbersome operation steps, expensive reagents and instruments, many facto

Method used

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  • Fluorescent polymerase chain reaction (PCR) kit for detecting CYP2C19 genotypes
  • Fluorescent polymerase chain reaction (PCR) kit for detecting CYP2C19 genotypes
  • Fluorescent polymerase chain reaction (PCR) kit for detecting CYP2C19 genotypes

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Experimental program
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Effect test

Embodiment 1

[0034] Embodiment 1: the preparation of kit

[0035] 1. Primer design and synthesis

[0036] Use Primer premier5.0 to design upstream and downstream amplification primers for the complete sequence of the CYP2C19 gene. The primers were synthesized by a professional company, and the primers were purified by PAGE.

[0037] The amplified sequence is shown in Table 1:

[0038] Table 1. Specific amplification primer sequences

[0039]

[0040] 2. Preparation of positive control substances for CYP2C19*2 and CYP2C19*3 genes

[0041] In this implementation, there are CYP2C19*2 type positive control substances 1-1, CYP2C19*2 type positive control substances 1-2, CYP2C19*2 type positive control substances 1-3, CYP2C19*3 type positive control substances 2-1, CYP2C19* Type 3 positive control substance 2-2, CYP2C19*3 type positive control substance 2-3.

[0042] CYP2C19*2 type positive control substance 1-1 is inserted with the wild homozygous recombinant plasmid with the nucleotide...

Embodiment 2

[0067] Embodiment 2: the use of kit

[0068] 1. Sample extraction and DNA preparation

[0069] 1) A blood sample is drawn from the patient to be tested to obtain the blood to be tested.

[0070] 2) Obtain DNA from the blood sample, and use a professional company's DNA extraction kit (purchased from Invitrogen) for extraction. Take 2.0 μl of the extract as a PCR reaction template.

[0071] 2. Preparation of positive control substances for CYP2C19*2 and CYP2C19*3 genes

[0072] According to the site to be detected in the sample, take the corresponding CYP2C19*2 positive control substance and CYP2C19*3 positive control substance in this kit.

[0073] 3. Preparation of CYP2C19*2 and CYP2C19*3 gene negative control substances

[0074] Take the β-actin plasmid provided by this kit as a negative control.

[0075] 4. Sample detection

[0076] The nucleic acid extracted in step 1, the positive control substance obtained in step 2, and the negative control substance obtained in st...

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Abstract

The invention provides a fluorescent polymerase chain reaction (PCR) kit for detecting CYP2C19 genotypes, and belongs to the field of in-vitro nucleic acid testing. The fluorescent PCR kit comprises PCR reaction liquids for detecting CYP2C19*2 and CYP2C19*3 genotypes, Taq DNA polymerase, and uracil-DNA glycosylase, wherein the PCR reaction liquids for detecting the CYP2C19*2 and CYP2C19*3 genotypes respectively comprise PCR amplification primers, minor groove binder (MGB) probes and the like; and nucleotide sequences for detecting the CYP2C19*2 and CYP2C19*3 genotypes are shown as SEQ ID NO:3-4 and SEQ ID NO:5-6 respectively. The kit has high sensitivity and specificity, can monitor the reaction progress in real time, ensures short reaction time, avoids subsequent treatment, can avoid reaction product pollution to the greatest extent, and can replace the traditional protein detection or the common PCR detection to diagnose the CYP2C19 genotypes.

Description

technical field [0001] The present invention relates to the field of in vitro nucleic acid detection, in particular to a genotype of two sites of the cytochrome oxidase P4502C19 (CYP2C19) gene that is commonly used in drug metabolism in clinical samples, namely CYP2C19*2 (681G / A) and CYP2C19 *3 (636G / A) fluorescent PCR kit. Background technique [0002] Cytochrome P450 (Cytochrome P450, CYP450) is an isoenzyme encoded by a group of structurally and functionally related superfamily genes (Superfamily gene). In the human body, it mainly exists in liver cell particles. In the CYP450 superfamily, CYP2 is the largest family with 15 subfamilies, which metabolize about 20% of the drugs currently used clinically. Among them, CYP2C19 (S-mephenytoin hydroxylase) is one of the main components of CYP450. The full length of CYP2C19 gene cDNA is 1940, contains 9 exons, and the coding region is 1473bp. The encoded protein S-mephenytoin hydroxylase metabolizes a series of clinical drugs, ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 黄双
Owner CHANGSHA 3G BIOTECH
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