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Primers, probe and kit for detecting ALDH2 (aldehyde dehydrogenase gene2) gene polymorphism

A gene polymorphism, kit technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial determination/inspection, etc., can solve cross-contamination of PCR products, insufficient enzyme digestion, instrument temperature control, etc. problem, to achieve the effect of good amplification effect, low synthesis cost and pollution reduction.

Inactive Publication Date: 2016-03-30
武汉海吉力生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there are many methods for the detection of ALDH2 gene polymorphisms. The simplest method is PCR-RFLP, but this method is complicated to operate, and when there are many samples, it is easy to cause cross-contamination of PCR products and prone to insufficient or excessive digestion. And false negative or false positive results, low reliability
Although the DNA sequencing method is the gold standard, the steps are cumbersome, the process is complicated, and cross-contamination between samples is prone to occur, resulting in sequencing failure. In addition, the price of the sequencer is beyond the tolerance of general clinical testing laboratories.
High-resolution melting curve method is fast, simple, economical and practical, but it is controlled by the temperature of the instrument, and the false positive is high

Method used

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  • Primers, probe and kit for detecting ALDH2 (aldehyde dehydrogenase gene2) gene polymorphism
  • Primers, probe and kit for detecting ALDH2 (aldehyde dehydrogenase gene2) gene polymorphism
  • Primers, probe and kit for detecting ALDH2 (aldehyde dehydrogenase gene2) gene polymorphism

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Embodiment 1 kit composition

[0067] The detection kit of the present invention is composed of an ALDH28-linked PCR reaction strip and an ALDH2 positive control group, and is divided into two types (see Table 1 and Table 2). The kit has 6 or 12 identical ALDH28-linked PCR reaction strips; the detection system corresponding to the odd-numbered tubes (1 / 3 / 5 / 7 or A / C / E / G) of each ALDH28-linked PCR reaction strip is A1 reaction solution , to detect ALDH2G1510A wild type; the detection system corresponding to even-numbered tubes (2 / 4 / 6 / 8 or B / D / F / H) is A2 reaction solution, to detect ALDH2G1510A mutant type (see Table 2). The main components of each tube are specific primers, fluorescent probes and PCR reaction solution, etc., in which the signal types of fluorescent probes are FAM signal and internal control JOE signal (internal control is used as the quality control of the kit, DNA quality and operation itself). Components in kits with different batch numbers are not int...

Embodiment 2

[0073] The extraction of embodiment 2 clinical sample DNA

[0074] 1. EDTA anticoagulant

[0075] In this example, genomic DNA was extracted from EDTA anticoagulated blood and quantified as a template for PCR detection. The blood genomic DNA extraction kit from QIAGEN Company was used, and the specific details are as follows.

[0076] 1. Processing blood material:

[0077] a. When the volume of the blood sample is less than 200 μL, add buffer GS to make up the volume to 200 μL, and then proceed to the next step of the experiment (if the volume of the blood sample is 200 μL, the next step of the experiment can be carried out directly without adding GS).

[0078] b. When the volume of the blood sample exceeds 200μL, it needs to be treated with cell lysate CL. The specific steps are as follows: add 1-2.5 times the volume of cell lysate CL to the sample, mix it upside down, and centrifuge at 10,000rpm (~11,500×g) After 1 min, remove the supernatant and leave the cell nucleus pe...

Embodiment 3

[0105] The extracted DNA was quantified with a UV spectrophotometer, and the DNA concentration was diluted to 1 ng / μL. Embodiment 3 Real-time fluorescent PCR method amplifies clinical sample DNA

[0106] This example uses the primers and probes provided by the present invention to amplify the DNA sample extracted in Example 1.

[0107] The fluorescent quantitative PCR reaction system for detecting ALDH2 gene polymorphism is:

[0108] PCR reaction solution:

[0109] PCR buffer (10×PCRBuffer (Mg 2+ Plus)): 2 ~ 8μL

[0110] dNTPs: 0.2~0.8mmol

[0111] Each primer: 0.1~1.0μmol

[0112] Each probe: 0.1~1.0μmol

[0113] Fastmasterpremix: 6-10 μL

[0114] Template: 10 μL

[0115] Total volume: 40 μL.

[0116] The preferred PCR reaction conditions are:

[0117] The first stage: 95°C for 5 minutes;

[0118] The second stage: 95°C for 5s, 58°C for 30s, 10 cycles;

[0119] The third stage: 95°C for 5s, 58°C for 30s, 72°C for 30s, 35 cycles;

[0120] Phase III: At 35 cycles, ...

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Abstract

The invention discloses primers, a probe and a kit for detecting ALDH2 (aldehyde dehydrogenase gene2) gene polymorphism and belongs to the technical field of gene polymorphism site detection. Nucleotide sequences of the primers comprise a mutant type ARMS upstream primer represented as SEQ ID NO:1, a wild type ARMS upstream primer represented as SEQ ID NO:2 and a common downstream primer represented as SEQ ID NO:3, and the probe adopts a common Taqman probe. The kit comprises an amplification primer of an internal control gene and the Taqman probe. The primers and the probe as well as the kit comprising the primers and the probe can be used clinically for detecting various types of samples and have the remarkable advantages of high specificity, high sensitivity, short experimental period, simplicity in operation, safety, non-toxicity, low cost and the like.

Description

technical field [0001] The invention relates to the technical field of gene polymorphism detection, in particular to primers, probes and kits for detecting ALDH2 gene polymorphism. Background technique [0002] Human acetaldehyde dehydrogenase (ALDH) is a tetrameric protein that catalyzes the oxidation of acetaldehyde and other aliphatic aldehydes. At present, 19 ALDH genes have been found, and among its 19 isoenzymes, they exist in liver mitochondria The ALDH2 activity in the cell is the strongest. [0003] ALDH2 has the activity of acetaldehyde dehydrogenase, which is a key enzyme in the metabolism of alcohol in the human body. It can convert the harmful product acetaldehyde into harmless acetic acid, thereby playing a role in detoxification. ALDH2 also has nitrate esterase activity, and nitroglycerin, a commonly used drug for clinical treatment and prevention of coronary heart disease angina pectoris, is also metabolized by it. Nitroglycerin is metabolized by ALDH2 to re...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68
CPCC12Q1/6886C12Q1/6858C12Q2600/112C12Q2600/156C12Q2561/113C12Q2561/101C12Q2545/101
Inventor 胡利红吴志伟夏琦段卫涛赵平锋
Owner 武汉海吉力生物科技有限公司
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