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Method of detecting apolipoprotein E gene type and kit

A technology of apolipoprotein and genotype, which is applied in the direction of biochemical equipment and methods, microbiological determination/inspection, etc., can solve the problems of high cost, insufficient resolution, complicated technology, etc., and achieve good detection stability and accurate and reliable results Effect

Inactive Publication Date: 2006-06-14
广州达安临床检验中心有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

These methods have the disadvantages of technical complexity, high cost, low efficiency and insufficient resolution.

Method used

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  • Method of detecting apolipoprotein E gene type and kit
  • Method of detecting apolipoprotein E gene type and kit
  • Method of detecting apolipoprotein E gene type and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Embodiment 1: Preparation of sample target nucleic acid

[0080] Draw 1ml of the subject's venous blood with a sterile syringe, add it to an anticoagulant tube containing EDTA, and store it at room temperature or at low temperature. Take 300 μl of anticoagulated whole blood and add it to a 1.5ml centrifuge tube, add 1ml of sterilized pure water to the centrifuge tube, mix well, and let stand at room temperature for 2-4 minutes. Then centrifuge (5000 rpm) for 6 minutes to collect the precipitate. After repeating the above steps once, add 1ml of normal saline to the centrifuge tube and mix well, centrifuge at 10000rpm for 10 minutes, and collect the white precipitate at the bottom of the tube. Add 50 μl of DNA extraction solution to the precipitate, mix thoroughly, bathe in boiling water for 10 minutes and centrifuge at 12000 rpm for 10 minutes. Take 2 μl of the supernatant as a template for the PCR reaction.

Embodiment 2

[0081] Embodiment 2: PCR amplification of target nucleic acid

[0082] Take several tubes of PCR reaction solution for one person, add 1 μl UDG enzyme to each tube, and then directly add 2 μl template (or negative and positive standards), mix well, and centrifuge instantaneously (3 seconds), and put each reaction tube into the PCR reaction tube. After pretreatment at 50°C for 3 minutes, hot start combined with "drop-down PCR" was used to amplify according to the following conditions: 94°C for 4 minutes, 80°C for 3 minutes, during which 1 μl of Taq enzyme was added. Denaturation at 94°C for 2 minutes, followed by 94°C for 1 minute, 70°C for 1 minute, 72°C for 1 minute, a total of 5 cycles, and then 94°C for 1 minute, 64°C for 1 minute, 72°C for 1 minute, a total of 30 cycles, A final 72°C extension was performed for 7 minutes. The amplified products were detected by 2% agarose gel electrophoresis (see attached figure 2 ).

Embodiment 3

[0083] Example 3: Sample reverse dot blot detection of six known genotypes

[0084] Before hybridization, hybridization solution I (2×SSC-0.1% SDS) was mixed with hybridization solution II and preheated to 59°C for use. According to the number of samples to be tested, take six 1.5ml centrifuge tubes, add 0.5ml hybridization solution I to each tube and preheat to 59°C. After the PCR amplification product was denatured at 97°C for 5 minutes, it was placed in an ice-water mixture for 2-5 minutes. Then take 1000 μl hybridization solution I+2 μl solution I (1000:2) mixed solution as the binding solution and store it at 4°C for later use, and take the mixture of solution II: solution III: solution IV (1900:200:1) (19ml solution II+2ml Solution III + 10 μl solution IV) was used as a chromogenic solution protected from light for later use.

[0085] Before hybridization, fill the reaction chamber with distilled water, place the metal perforated plate, and turn on the water pump to dr...

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Abstract

The invention relates to examination clinic blood sample ApoE gene subtype method and kit, especially reverse dot blot hybridization technique fast measuring ApoE gene type and its kit.

Description

[0001] Field [0002] The invention relates to a method and kit for detecting apolipoprotein (ApoE) genotyping in clinical blood samples, in particular to a method for rapidly detecting ApoE genotype by reverse dot hybridization technology and the used kit. Background of the invention [0003] The human ApoE gene has three alleles, namely ε2, ε3 and ε4, which constitute 6 different genotypes: 3 homozygous types (ε4 / ε4, ε3 / ε3 and ε2 / ε2) and 3 heterozygous types ( ε3 / ε4, ε2 / ε3 and ε2 / ε4). The allele frequency varies among different races and regions. In the general population, the frequencies of ε2, ε3 and ε4 alleles are 8%, 78% and 4%, respectively. [0004] Currently known, ApoEε4 is an independent risk factor for premature coronary heart disease (coronary heart disease, CHD). Y, Stampfer MJ, Liu S, et al. Meta-analysis: apolipoprotein E genotypes and risk for coronary heart disease. Ann Intern Med. 2004, 141(2): 137-147.). A large number of studies in recent years have fou...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 王伟毅张太松何蕴韶程钢汪华侨
Owner 广州达安临床检验中心有限公司
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