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Method, primer and kit for detecting SLCO1B1 gene mutation

A technology of kits and sequencing primers, which is applied in the fields of life science and biology, can solve problems such as many operation steps, high price, and specificity impact, and achieve the effects of simple equipment requirements, easy promotion, and easy operation

Inactive Publication Date: 2018-07-24
南昌艾迪康医学检验实验室有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] 3) PCR high-resolution melting curve analysis technology can detect gene mutations in high throughput, and the reagent cost is low, but because the fluorescent signal comes from the dye, the specificity is affected, and the detection instrument is an upgraded version of the fluorescent PCR instrument. Expensive, limited popularity
[0008] 4) The PCR-Taqman MGB probe method is suitable for known mutation sites, and often requires two probes, and the synthesis price of TaqmanMGB probes is several times that of ordinary Taqman probes, and cannot detect samples with less content, etc. bit gene or mutation site
[0009] 5) Although the PCR-SSCP method is simple, it is an open detection system, which is easy to cause PCR product contamination, and there are many operating steps, which are time-consuming and labor-intensive

Method used

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  • Method, primer and kit for detecting SLCO1B1 gene mutation
  • Method, primer and kit for detecting SLCO1B1 gene mutation
  • Method, primer and kit for detecting SLCO1B1 gene mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Primers for detecting mutations in the SLCO1B1 gene, the primers are designed for exons 5 and 6 of the SLCO1B1 gene, and the primers include:

[0054] A pair of amplification primers targeting exon 5 of the SLCO1B1 gene, the base sequence of which is:

[0055] SLCO1B1-Exon5-F: TGTAAAACGACGGCCAGTTTATCTTTCTTGCTGGACACT

[0056] SLCO1B1-Exon5-R: AACAGCTATGACCATGTAATACAGTCATCCCTTGGTA;

[0057] A pair of amplification primers targeting exon 6 of the SLCO1B1 gene, the base sequence of which is:

[0058] SLCO1B1-Exon6-F: TGTAAAACGACGGCCAGTTTGCTCTTCCTTCATCTTCCG

[0059] SLCO1B1-Exon6-R: AACAGCTATGACCATGTTTCAAAAAGTAGACAAAGGGA.

[0060] In particular, the primers also include a pair of sequencing primers, and the base sequence of the sequencing primers is:

[0061] Sequencing Primer F: TGTAAAACGACGGCCAGT

[0062] Sequencing primer R: AACAGCTATGACCATG.

[0063] A kit for detecting SLCO1B1 gene mutation, comprising:

[0064] (1) detection system PCR reaction solution;

[006...

Embodiment 2

[0077] Example 2 sample DNA amplification

[0078] Operation process of blood / cell / tissue genomic DNA extraction kit (Tiangen Biology):

[0079] (1) Add 20 μl of QIAGEN Protease (or proteinase K) to the bottom of a 1.5ml centrifuge tube.

[0080] (2) Add 200 μl plasma to the centrifuge tube.

[0081] (3) Add 200μl Buffer AL and shake for 15s. (Note: Do not add QIAGEN Protease or Proteinase K directly to Buffer AL. If the sample size is large, increase QIAGEN Protease and Buffer AL proportionally.)

[0082] (4) Water bath at 56°C for 10 minutes, and then briefly centrifuge to remove the liquid on the inner edge of the centrifuge tube cover.

[0083] (5) Add 200 μl of ethanol (96%-100%), shake for 15 s, and centrifuge briefly to remove the liquid along the inner edge of the centrifuge tube cover.

[0084] (6) Carefully add the mixture obtained above (including the precipitate) into the QIAamp Mini spin column (without wetting the rim). Put the spin column into a 2ml collecti...

Embodiment 3

[0089] Example 3 sample DNA amplification

[0090] The blood sample DNA extracted according to Example 2 was then amplified using the amplification primers in Example 1 to amplify Exon 5 and Exon 6 of the SLCO1B1 gene. Amplification was carried out on a conventional PCR instrument, available instruments include ABI veriti (Applied Biosystems, USA) and the like. The details are as follows:

[0091](i) According to the number of samples n (number of samples = number of samples to be tested + 1 negative control + 1 positive control + 1 blank control), configure the PCR amplification reaction solution for the test system, and distribute 19 μL in each tube into reaction tubes;

[0092] (ii) Add 1 μL each of the above-mentioned processed sample to be tested, the negative control substance, and the positive control substance into the reaction tube, mix well, centrifuge at low speed for several seconds, perform PCR amplification, and obtain the amplified product. The preparation met...

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Abstract

The invention discloses a primer, a method and a kit for detecting SLCO1B1 gene mutation related to CHD (Coronary Atherosclerotic Heart Disease). The primer comprises two pairs of amplification primers aiming at a fifth exon sequence and a sixth exon sequence and a pair of sequencing primers; by adopting a Sanger sequencing technology, gene mutation related to the CHD can be rapidly detected. A result of detection completed by utilizing the primer, the method and the kit, which are disclosed by the invention, is accurate, and an important reference significance in early diagnosis and individualized treatment of the CHD is obtained.

Description

technical field [0001] The invention belongs to the field of life science and biotechnology, and particularly relates to a primer, a method and a kit for detecting SLCO1B1 gene mutation related to coronary heart disease. Background technique [0002] Coronary Atherosclerotic Heart Disease (CHD) is the most common cause of death worldwide, and elevated levels of low-density lipoprotein cholesterol (LDL-C) are the most important cause of CHD. Effectively lowering LDL-C levels can prevent the progression of atherosclerosis, thereby reducing the morbidity and mortality of CHD. Statins are currently the most effective drugs for reducing plasma LDL-C levels and thereby reducing the risk of cardiovascular diseases. Studies have shown that there are significant individual differences in the pharmacokinetics of statins, and this difference may affect the efficacy of statins and the occurrence of adverse reactions. This difference is also closely related to the SLCO1B1 genotype. Th...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q2535/101C12Q2531/113
Inventor 董绍斌吴鹏飞王淑一
Owner 南昌艾迪康医学检验实验室有限公司
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