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Primer set for detecting genotyping of acetaldehyde dehydrogenase 2, reagent, kit and detection method and application

An acetaldehyde dehydrogenase and genotyping technology, applied in the field of primer sets for detecting acetaldehyde dehydrogenase 2 genotyping, can solve the problems of long cycle and high development cost, achieve high accuracy, low development cost, Easy-to-use effects

Inactive Publication Date: 2018-12-11
QINGDAO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The purpose of the present invention is to provide a kind of primer group that is used for detecting aldehyde dehydrogenase 2 genotyping, to alleviate the Taqman probe-qPCR that exists in the prior art to detect acetaldehyde dehydrogenase 2 genotyping has development cost High and long-term technical problems

Method used

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  • Primer set for detecting genotyping of acetaldehyde dehydrogenase 2, reagent, kit and detection method and application
  • Primer set for detecting genotyping of acetaldehyde dehydrogenase 2, reagent, kit and detection method and application
  • Primer set for detecting genotyping of acetaldehyde dehydrogenase 2, reagent, kit and detection method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0081] Example 1 Positive reference sample genotyping analysis

[0082] After the analysis of the first-generation sequencing results, this example randomly selected three DNA samples with genotypes of GG, GA and AA as positive references, the DNA purity was between 1.7 and 1.8, and the concentration was 10 ng / μL. The provided kit uses the following detection method to detect the ALDH2 rs671 locus genotype of the positive reference sample:

[0083] (a) Site-specific PCR: according to the sample type, set a no-template control group (NTC) and a positive reference group. According to the kit instructions, 2*PCR Mix was melted and mixed for system configuration, and 5 μL of 2*PCR Mix was added to the reaction system, ddH 2 O 3 μL, Primer Mix 1 μL (primer final concentration 200pM), positive reference DNA sample 1 μL. The DNA samples of the NTC group were pure water, and the DNA samples of the positive reference group were three DNAs of genotypes GG, GA, and AA. The number of r...

Embodiment 2

[0088] Embodiment 2 large sample genotyping analysis

[0089] Randomly select 84 test subjects, extract whole blood DNA, and test the concentration and purity, and use the following detection method to detect the genotype of the ALDH2 rs671 locus of the sample to be tested:

[0090] (A) Whole blood DNA extraction: 400 μL of peripheral anticoagulated blood was extracted with a whole blood DNA extraction kit, the concentration was 1-100 ng / μL, and the OD260 / 280 was between 1.7 and 1.9.

[0091] (B) PCR at specific sites: according to the sample type, set a no-template control group (NTC), a positive reference group and an experimental group. According to the kit instructions, 2*PCR Mix was melted and mixed for system configuration, and 5 μL of 2*PCR Mix was added to the reaction system, ddH 2 O 3 μL, Primer Mix 1 μL (primer final concentration 200pM), DNA sample 1 μL. The DNA samples of the NTC group are pure water, the DNA samples of the positive reference group are three DNAs ...

Embodiment 3

[0097] Typing detection of embodiment 3 case samples

[0098] One case of whole blood DNA was extracted from a patient with angina pectoris, the concentration was 72ng / μL, and the purity (OD260 / 280) was 1.86. The ALDH2 rs671 site genotype of the patient was detected by the detection method provided in Example 2 of the present invention.

[0099] Experimental results such as Figure 4 As shown, there is no fluorescent signal in the NTC group, and the signal is in the lower left corner. The signals of the three positive reference products GG, GA, and AA are normal. The patient sample has a good typing result, and the result is GG type.

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Abstract

The invention provides a primer set for detecting genotyping of acetaldehyde dehydrogenase 2, a reagent, a kit, a detection method and application, and relates to the technical field of genetic engineering. The primer set for detecting genotyping of acetaldehyde dehydrogenase 2 comprises a common upstream primer, an allele-specific primer, and an intermediate sequence-specific primer. The primer set has the characteristics of low development cost, short development cycle, high accuracy and the like, and can meet the requirements of accuracy, precision and detection rate and the like required for clinical detection. Moreover, the primer set has the advantages of high specificity, high accuracy, and wide range of DNA measurement, and is favorable for promoting the clinical detection of the nitroglycerin individualized drug gene for the locus. The method for detecting genotyping of acetaldehyde dehydrogenase 2 provided by the invention has low detection cost, and the genotype of ALDH2 rs671 locus can be analyzed economically, accurately and rapidly by using the detection method provided by the invention, which is of great significance in clinical promotion of the nitroglycerin individualized drug gene detection.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a primer set, a reagent, a test kit, a detection method and an application for detecting the genotype of acetaldehyde dehydrogenase 2. Background technique [0002] Nitroglycerin (GTN, glyceryl trinitrate, nitroglycerin) has been used to treat angina pectoris and heart failure for 130 years. Sublingual administration of nitroglycerin tablets is the routine first-choice prescription for anti-angina pectoris acute attack of coronary heart disease, but its clinical effectiveness is often due to It varies from person to person. Some people saved their lives in time because of it, but after some people took it, the effect was not very obvious. In the past, the domestic and foreign medical circles have always believed that the poor curative effect of nitroglycerin on some patients is caused by its "drug resistance", but the research results prove that this is not the case....

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/6883C12Q2600/106C12Q2600/112
Inventor 李培峰李保伟刘英郝晓丹吴薇
Owner QINGDAO UNIV
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