Detection method and kit for coronary heart disease susceptibility loci rs1801133

A technology for coronary heart disease and a kit, applied in the fields of molecular biology and medicine, can solve the problems of affecting activity and heat resistance, reducing enzyme activity, reducing heat resistance, etc., and achieving accurate results, simple operation and low cost of use. Effect

Active Publication Date: 2014-09-24
光翰科技(上海)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

C677T is located in the catalytic region of MTHFR. This mutation directly aff

Method used

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  • Detection method and kit for coronary heart disease susceptibility loci rs1801133
  • Detection method and kit for coronary heart disease susceptibility loci rs1801133
  • Detection method and kit for coronary heart disease susceptibility loci rs1801133

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1: Fluorescent PCR detection

[0029] 1. Experimental materials

[0030] The LightCycler480 fluorescent quantitative PCR instrument was purchased from Roche, Switzerland, and the polymerase chain reaction solution (LightCycler480High Resolution Melting Master) was custom-synthesized by Roche, Switzerland.

[0031] 2. Primer and probe design and synthesis:

[0032]Using the partial sequence of the exon of the MTHFR gene as a template, the primers were analyzed using Primer Premier5 software and custom-synthesized by Shanghai Sangon Bioengineering Co., Ltd.

[0033] Primers for detection:

[0034] MTHFR gene rs1801133 upstream primer sequence: 5′-

[0035] ACCTGAAGCACTTGAAGGAGAA-3' (SEQ ID NO 2)

[0036] MTHFR gene rs1801133 downstream primer sequence: 5'-CAAAGCGGAAGAATGTGTCAG-3' (SEQ ID NO3)

[0037] 3. Sample testing:

[0038] A total of 50 cases of coronary heart disease and 50 normal controls were detected in the experiment. About 2ml of blood samples...

Embodiment 2

[0046] Example 2: Detection of the genotype of the coronary heart disease rs1801133 locus by Taqman method

[0047] The rs1801133 site of MTHFR gene, a genetic risk gene for coronary heart disease, was detected by Taqman method. Select 10 samples from each of the above coronary heart disease cases and control groups for Taqman typing test to determine the genotype of rs1801133.

[0048] 1. Experimental method

[0049] The Taqman probe method uses the Taqman probe of Applied Biosystems and the Taqman Mix of Suzhou Xinhai Biotechnology Co., Ltd.

[0050] Carry out PCR amplification according to the following system, and finally use Roche LC480software1.5 to scan and cluster analysis

[0051]

384-well plate (ul)

Taqman Mix

5

Taqman Probe

0.1

DNA (about 5ng / μl)

1

H2O

3.9

total reaction volume

10

[0052]

[0053] 2. Experimental results

[0054] The screenshot of the Taqman typing result is as follow...

Embodiment 3

[0059] Example 3: Detection Kit for Coronary Heart Disease Susceptibility Loci

[0060] The detection kit provided by the present invention can amplify and analyze the region containing the rs1801133 site in the MTHFR gene exon, and the detection kit can detect the susceptibility of coronary heart disease, and it contains the MTHFR gene that can be amplified The exon contains specific primers for the rs1801133 site, and other PCR-HRM corresponding reagents, and the PCR amplification and HRM analysis components have the same content.

[0061] The detection kit is for 50 people for detection and application, and it is stored at -20°C in the dark. Its components and contents include:

[0062] 5ul2X LightCycler480High Resolution Melting Master Mix

[0063] 1.2ul 25mM MgCl2 solution

[0064] 1ul 2μM Primer Mix

[0065] About 1ul5ng / μl DNA

[0066] 1.8ul H2O

[0067] The specific primers having the nucleotide sequences shown in SEQ ID NO: 2 and SEQ ID NO: 3 were 0.2 uM each.

...

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Abstract

The invention relates to a detection method and a kit for coronary heart disease susceptibility loci. The method comprises the following steps: (1) extracting a genome deoxyribonucleic acid (DNA) of a sample, and amplifying a region containing rs1801133 loci in a methylene tetrahydrofolate reductase (MTHFR) gene exon of the sample, so as to obtain an amplification product; (2) detecting the genetype of single nucleotide polymorphism (SNP) loci rs1801133 in the amplification product by using a high resolution melting (HRM) analysis technology. The genetype analysis is carried out on the region containing the rs1801133 loci in the amplified MTHFR gene by adopting the HRM analysis technology. The method is simple and quick to operate, low in use cost and accurate in result, and can achieve batch inspection; whether the tested people carry with coronary heart disease susceptibility genes can be clearly judged by using the kit, the coronary heart disease susceptibility people can be screened out from people, the poor living habit is improved, and the target of prevention is achieved.

Description

【Technical field】 [0001] The present invention relates to a method and a kit for detecting the susceptibility of coronary heart disease, more specifically by measuring the single nucleotide polymorphism (single nucleotide polymorphism) of the gene methylenetetrahydrofolate reductase (MTHFR) related to coronary heart disease. Nucleotide polymorphism, SNP) predicts the subject's susceptibility to coronary heart disease. This method can be used for auxiliary diagnosis, treatment and new drug development of the disease, and belongs to the field of molecular biology and medicine. 【Background technique】 [0002] Coronary heart disease is a heart disease caused by atherosclerotic lesions in the coronary arteries that cause stenosis or blockage of the vessel lumen, resulting in myocardial ischemia, hypoxia or necrosis. A large number of epidemiological investigations have shown that elevated plasma homocysteine ​​levels are associated with increased risk of atherosclerotic vascular ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6858C12Q1/6883C12Q2600/156C12Q2531/113C12Q2527/107
Inventor 张天竹黄迅威沈玲宇邵敏华
Owner 光翰科技(上海)有限公司
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