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Human ALDH2 gene polymorphism detection kit as well as preparation method and application thereof

A detection kit and gene technology, which can be used in biochemical equipment and methods, recombinant DNA technology, and microbial determination/inspection, etc., can solve the problems of long experiment time, low sensitivity of PCR-sequencing method, and unsuitable detection of mutation frequency. , to prevent false negative and false positive results, improve specificity and accuracy, and interpret the results easily and objectively

Active Publication Date: 2018-12-21
WUHAN HEALTHCHART BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The PCR-sequencing method has low sensitivity and takes a long time for the experiment, so it is not suitable for clinical promotion; the chip hybridization method has low detection sensitivity and poor specificity, and is prone to false positive results; the high-resolution melting curve method has special requirements for equipment and is not suitable for clinical promotion. Detection sensitivity is not high
The Taqman-qPCR method has high detection sensitivity, but often due to the site sequence, the detection specificity is not high, and it is genotyped by the Genotyping method, which has certain requirements for the number of samples and polymorphism distribution, and is not suitable for detecting mutation frequency too low position

Method used

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  • Human ALDH2 gene polymorphism detection kit as well as preparation method and application thereof
  • Human ALDH2 gene polymorphism detection kit as well as preparation method and application thereof
  • Human ALDH2 gene polymorphism detection kit as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1 prepares ALDH2 gene detection kit of the present invention

[0049] 1. Primer design and synthesis:

[0050] The ALDH2 1510 site reaction system contains 6 primers; two sense chain upstream primers F1 and F2, two antisense chain downstream primers R1 and R2, F1 and R1 are common primers, F2 and R2 are fluorophore and The specific ARMs primers of the tag sequence are screened by primers and PCR reaction conditions to ensure that the primer pair F1R1 enriches the template, while F2 and R1, F1 and R2 can normally amplify specific genotype PCR products with fluorescence; at the same time, Internal reference primers were added to the gene locus.

[0051] The specific sequence is as follows:

[0052] ALDH2-F1: 5'-TGTTTGGAGCCCAGTCACCC-3' SEQ ID NO.1

[0053] ALDH2-F2: 5'-FAM-AAGATACATTGATGACATACACT A -3’ SEQ ID NO.2

[0054] (Specific recognition of mutation template)

[0055] ALDH2-R1: 5'-ACCAGCAGACCCTCAAGCCC-3' SEQ ID NO.3

[0056] ALDH2-R2: 5'-VIC-ATCCCT...

Embodiment 2

[0081] The human ALDH2 gene polymorphism detection kit prepared in Example 1 was used to detect the samples to be tested. In this example, 100 samples of EDTA anticoagulated venous whole blood were collected, genomic DNA was extracted, and the human ALDH2 gene polymorphism detection kit was used to detect the ALDH2 1510 gene polymorphism. The specific operation process was as follows:

[0082] (1) Genomic DNA extraction from blood samples: Use a commercial extraction kit to extract genomic DNA. After the extraction is complete, use TE buffer to elute the DNA and measure the DNA concentration; dilute the genomic DNA to 20ng / μl;

[0083] (2) Fluorescence quantitative detection: Add 30 μl of PCR premixed reaction solution and 20 μl of sample DNA to be tested into the reaction tube; the PCR reaction program is: 95°C for 1 minute pre-denaturation; 15 cycles: 95°C for 5 seconds, 61°C for 32 seconds , do not collect fluorescence; 30 cycles: 95°C for 5 seconds, 61°C for 32 seconds, co...

Embodiment 3

[0094] Use the kit prepared in Example 1 to detect and verify the minimum detection line of this kit, specifically including the following steps:

[0095] (1) Using the samples of known ALDH2 corresponding site genotypes in Example 2, select one case of wild-type genome, mutant genome and heterozygous genome samples for each site, and dilute the above samples to 10ng / μL, 1ng / μL, 0.5ng / μL, 0.25ng / μL, 0.1ng / μL, 0.05ng / μL, 0.025ng / μL, 0.001ng / μL;

[0096] (2) Fluorescence quantitative detection: Add 30 μl of PCR premixed reaction solution and 20 μl of sample DNA to be tested into the reaction tube; the PCR reaction program is: 95°C for 1 minute pre-denaturation; 15 cycles: 95°C for 5 seconds, 61°C for 32 seconds , do not collect fluorescence; 30 cycles: 95°C for 5 seconds, 61°C for 32 seconds, collect fluorescence; each concentration gradient of each sample is repeated 20 times;

[0097] (3) After the PCR reaction is completed, perform result interpretation according to Table 2;...

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Abstract

The invention belongs to the technical field of biology and specifically relates to a human ALDH2 gene polymorphism detection kit as well as a preparation method and application thereof. The kit is prepared from PCR premixed reaction liquid, a positive control product and a negative control product, wherein the PCR premixed reaction liquid is used for amplifying a G1510A site of ALDH2 gene; the PCR premixed reaction liquid is prepared from a specificity primer sequence set, a probe set and PCR reaction liquid; the specificity primer sequence set is used for amplifying all the sites; the specificity primer set is prepared from an ordinary outer primer and an ARMs primer with fluorescent label specificity. The kit disclosed by the invention is used for detecting the human ALDH2 gene polymorphism and has the advantages of high flexibility, high specificity, convenience in operation, reliable results and the like; furthermore, detection can be finished within 1 hour, and result reading issimple and objective.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a human ALDH2 gene polymorphism detection kit and its preparation method and application. Background technique [0002] The human aldehyde dehydrogenase 2 (aldehyde dehydrogenase, ALDH2) gene is located on chromosome 12, contains 13 exons, and encodes two isomerases of acetaldehyde dehydrogenase and nitrate esterase. ALDH2 is an NAD(P)+-dependent enzyme, which is widely involved in the oxidation reaction of aldehydes produced during the metabolism of ethanol, amino acids, biogenic amines, vitamins, cholesterol and lipids in the body, and becomes the corresponding carboxylic acid, which is beneficial for alleviating Aldehydes play an important role in the toxicity of the body. Many mutants such as His47Arg, Glu479Lys, and Glu504Lys have been found so far, among which the main mutant is Glu504Lys (rs671), which is the single-base mutation G1510A located in exon 12. The no...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/6858
CPCC12Q1/6858C12Q2537/143C12Q2563/107C12Q2561/101C12Q2545/113
Inventor 王宁付金玲李倩李雪梅叶伦程弘夏陈刚
Owner WUHAN HEALTHCHART BIOLOGICAL TECH
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