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Nitroglycerin metabolism marker detection kit and detection method and application thereof

A technology for detecting kits and metabolic markers, which is applied in biochemical equipment and methods, microbe determination/inspection, DNA/RNA fragments, etc. It can solve the problems of easy pollution of amplification products, high test cost, and strict detection conditions And other issues

Pending Publication Date: 2021-11-02
湖南菲思特精准医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the sequencing method and the chip method have cumbersome operation steps, long detection cycle, and the amplification product is prone to contamination; the high-resolution melting curve method has simple steps, low specificity, and high requirements for equipment; The specific amplification method uses ARMS primers for specific amplification, and the design of the primers is difficult to optimize, and the detection conditions are strict.
Taqman fluorescent probe method has high test cost, and the amplification throughput of multiple genes is not high

Method used

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  • Nitroglycerin metabolism marker detection kit and detection method and application thereof
  • Nitroglycerin metabolism marker detection kit and detection method and application thereof
  • Nitroglycerin metabolism marker detection kit and detection method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1, the preparation of kit

[0036] The kit of the present invention designs specific amplification primers and sequencing primers for ALDH2 (G1510A), and is used for constant temperature amplification and pyrosequencing detection. The design of primers based on the recombinant enzyme polymerase amplification technology is one of the keys of the present invention. The primer design of this technology cannot be carried out by auxiliary software, and can only rely on manual design. In order to ensure the amplification speed and detection sensitivity, the primer length should be controlled at 30-35 bp. If the primer design is too short, non-specific amplification will easily increase and cause false positives. If the primer design is too long, it will easily lead to failure of amplification. Gene polymorphism sequence is subject to the public sequence in Genebank.

[0037] (1) The primer sequences of this embodiment are as follows:

[0038]

[0039] (2) The de...

Embodiment 2

[0048] Embodiment 2, pyrophosphate detection

[0049] The instrument that adopts among the present invention is as follows: thermostat;

[0050] Pyrosequencer: Wuhan First Biotechnology Co., Ltd.

[0051] (1) Reagent preparation (reagent preparation room)

[0052] Take out the reagent in advance, vortex reagent 1 for 15 seconds, and centrifuge at low speed for later use. Add 440ul of Reagent 1 directly to Reagent 2 (lyophilized), and vortex for 15 seconds to mix well. Determine the number of reactions N, N = number of samples to be tested (n) + number of quality control products (1) + blank control. It is recommended to conduct positive control and blank control analysis for each PCR experiment at the same time. Then the reaction solution was dispensed into PCR reaction tubes at 20 μL / tube.

[0053] (2) Sample testing (sample preparation room)

[0054] Add the sample DNA, positive control and blank control into the PCR reaction tube according to the amount of 5 μL, close...

Embodiment 3

[0074] Example 3. Correlation between genetic testing results and drug efficacy

[0075] Using the blood samples of 96 patients taking sublingual nitroglycerin drugs, ALDH2 was genotyped, and the correlation between drug efficacy and genotype was analyzed. The results showed that the effective rate of ALDH2 wild-type sublingual nitroglycerin was significantly higher than that of the genotype containing the *2 allele.

[0076] The efficacy of nitroglycerin ALDH2*1 / *1 type ALDH2*1 / *2, *1 / *2 type Nitroglycerin active group 43(86％) 18(39.13％) Nitroglycerin ineffective group 7(14％) 28(60.87％)

[0077] The genotypes corresponding to ALDH2 are ALDH2*1 / 1, ALDH2*1 / 2, and ALDH2*2 / 2. The catalytic activity of the mutant type towards nitroglycerin was significantly lower than that of the wild type. The correlation between ALDH2 genotype and nitroglycerin drug efficacy is summarized as follows:

[0078]

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Abstract

The invention discloses a nitroglycerin metabolism marker detection kit and a detection method and application thereof. The detection kit is used for detecting the gene polymorphism of a nitroglycerin metabolism marker gene locus ALDH2G1510A, and the kit comprises an ALDH2G1510A amplification primer, an ALDH2G1510A sequencing primer and a positive control. According to the method, ALDH2 (G1510A) is amplified through RPA, a large number of amplification products are rapidly and effectively generated at the constant temperature, single-stranded DNA is specifically captured through an amino-labeled single-stranded DNA analogue directly combined with carboxyl modifier, after washing, a sequencing primer and template DNA are added for annealing, and a sequencing raw material is added for pyrosequencing.

Description

technical field [0001] The invention relates to a detection kit for a nitroglycerin metabolic marker, a detection method and an application thereof, and belongs to the field of gene detection. Background technique [0002] Nitroglycerin is the first-line treatment drug for angina pectoris and acute myocardial infarction, which can effectively relieve the symptoms of angina pectoris. The ineffective rate of Chinese people taking nitroglycerin is as high as 25%. The clinical effectiveness of nitroglycerin often varies from person to person. When some patients have angina pectoris, nitroglycerin will be ineffective, or it will take more than half an hour to be effective. Studies have shown that ALDH2 gene mutation is the decisive factor affecting the efficacy of nitroglycerin. The ALDH2 gene is located on human chromosome 12. ALDH2 has both acetaldehyde dehydrogenase and esterase activities, and participates in the metabolism of ethanol, nitroglycerin and other drugs. ALDH2 m...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/6844C12Q1/6869C12N15/11
CPCC12Q1/6883C12Q1/6844C12Q1/6869C12Q2600/106C12Q2600/156C12Q2521/507C12Q2522/101C12Q2537/143C12Q2565/301
Inventor 陈立波刘丹
Owner 湖南菲思特精准医疗科技有限公司
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