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Composition, kit and method for detecting human ethanol metabolism gene polymorphism

A technology for ethanol metabolism and composition, which is applied in the fields of biochemical equipment and methods, recombinant DNA technology, and microbial determination/inspection, which can solve the problem that it cannot be applied to whole blood samples and oral swab samples at the same time, and the PCR amplification process takes a long time. , the sample adaptation range is narrow and other problems, to avoid mutual interference and superposition, facilitate PCR amplification operation, and achieve the effect of high accuracy

Active Publication Date: 2020-02-04
SANSURE BIOTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there are some kits based on PCR technology for detecting ethanol metabolism; however, one or more of the following defects restrict the application of ethanol metabolism detection reagents: 1) Genotyping can only be interpreted based on PCR-gel electrophoresis band analysis or melting curve analysis, so professionals are required to use it; 2) The scope of sample adaptation is narrow and cannot be applied to whole blood samples and oral swab samples at the same time ; 3) The PCR amplification process takes a long time; 4) The detection accuracy is low

Method used

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  • Composition, kit and method for detecting human ethanol metabolism gene polymorphism
  • Composition, kit and method for detecting human ethanol metabolism gene polymorphism
  • Composition, kit and method for detecting human ethanol metabolism gene polymorphism

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Embodiment 1, primers and probes used in the present invention

[0054] Aiming at the 150 bp before and after exon rs671 (G1510A) of exon 12 of ALDH2 gene and exon 3 of ADH1B gene rs1229984 (A143G) as the target region, the sequences of primers and probes of ALDH2 gene were determined as follows:

[0055] ALDH2-F: 5'-AGTCACCCCTTTGGTGGCTACAAGAT-3' (SEQ ID NO: 1);

[0056] ALDH2-R:5'-GCTCCGAGCCACCAGCAGA-3' (SEQ ID NO:2);

[0057] ALDH2-1510G-P:5'-AGTTTTCACTTCAGTGTAT-3' (SEQ ID NO:3);

[0058] ALDH2-1510A-P: 5'-CAGTTTTCACTTTAGTGTAT-3' (SEQ ID NO: 4);

[0059] Determine the sequences of ADH1B gene primers and probes as follows:

[0060] ADH1B-F:5'-CAGGAATTTGGGTATGTTAAATTCATC-3' (SEQ ID NO:5);

[0061] ADH1B-R: 5'-ACCAG GTTGCCACTAACCACGT-3' (SEQ ID NO: 6);

[0062] ADH1B-143A-P:5'-CGTGGTCATCTGTGTGACA-3' (SEQ ID NO:7);

[0063] ADH1B-143G-P: 5'-TGGTCATCTGTGCGACAG-3' (SEQ ID NO: 8).

[0064] Among them, the fluorescent reporter group of ALDH2-1510G-P is FAM; the fluores...

Embodiment 2

[0065] Embodiment 2, the method for detecting ethanol metabolism capacity

[0066] 1 Reagent preparation:

[0067] According to the number of samples to be tested, ALDH2 / ADH1B negative control, and ALDH2 / ADH1B positive control, take the corresponding amount of PCR reaction solution and enzyme in proportion (reaction solution 16-20 μL / person + enzyme mixture 1-2 μL / person) Mix the solution thoroughly to form a PCR-mix, centrifuge briefly and set aside.

[0068] The PCR reaction solution is 5 μL of 10 × PCR reaction buffer, 2.5 mmol / L deoxyribonucleoside triphosphate, 0.2 μmol / L for two sets of upstream and downstream primers for target polynucleotide amplification, 0.2 μmol / L for two The probe of polymorphic site detection, described 10 * PCR reaction buffer solution comprises the 200mmol / L tris hydrochloride solution of pH 7.5, 30mmol / L magnesium chloride solution, 500mmol / L potassium chloride solution, 0.2% (v / v) triton solution and 10% (v / v) formamide solution.

[0069] 2...

Embodiment 3

[0084] Embodiment 3, detection result and next-generation sequencing result comparative test

[0085] According to the experimental program provided by the present invention, 27 peripheral blood samples and 23 oral swab samples with unknown detection results were tested, and compared with the next-generation sequencing test results, the results are as follows:

[0086]

[0087]

[0088]

[0089] Conclusion: The present invention can accurately detect various genotypes of peripheral blood and oral swab samples, and the sequencing results of ALDH2 gene exon 12 rs671 (G1510A) and ADH1B gene exon 3 rs1229984 (A143G) are consistent with those of human ALDH2 gene and The detection results of the ADH1B gene polymorphism nucleic acid detection kit in the rapid fluorescent quantitative PCR instrument are completely consistent.

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Abstract

The invention provides a composition for detecting ethanol metabolic capacity, and relates to the field of molecular biology detection, in particular to the field of ethanol metabolic capacity detecting. Meanwhile, the invention further provides application of the composition to detection of the ethanol metabolic capacity, a kit comprising the composition, and a method for detecting the ethanol metabolic capacity. By using the composition and the method, the detection time can be greatly shortened.

Description

technical field [0001] The invention belongs to the field of molecular biology detection, more specifically, to the field of polymorphism detection of human ethanol metabolism gene. Background technique [0002] The chemical name of alcohol is ethanol. After drinking, ethanol is absorbed into the blood through the stomach, 90% to 95% of the ethanol is metabolized by the liver, and the rest is excreted with urine, sweat and breath. Ethanol mainly undergoes two steps of metabolic reactions in the liver: the oxidation of acetaldehyde by alcohol dehydrogenase ADH1B; and the oxidation of acetaldehyde to acetic acid by acetaldehyde dehydrogenase. Alcohol dehydrogenase is mainly encoded by the ADH1B gene, and acetaldehyde dehydrogenase is mainly encoded by the ALDH2 gene. In the process of alcohol metabolism, people with high activity of acetaldehyde dehydrogenase have strong ability to metabolize alcohol; people with low activity of acetaldehyde dehydrogenase have weak ability to...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/6858C12N15/11
CPCC12Q1/6888C12Q1/6858C12Q2600/156C12Q2531/113C12Q2563/107
Inventor 戴立忠缪为民龙凤英吴康
Owner SANSURE BIOTECH INC
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