Establishment of methodology for detecting genes affecting efficacy of lipid-lowering drugs by TaqMan-MGB probe technique

A probe and gene technology, applied in the field of TaqMan-MGB probe technology to detect genes that affect the efficacy of lipid-lowering drugs, can solve the problems of increasing the incidence of severe myotoxicity, shorten the detection cycle, and reduce the risk of false positives , high resolution effect

Inactive Publication Date: 2019-06-18
江苏百世诺医疗科技有限公司
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Problems solved by technology

When used in combination, drug interactions may occur, which can increase the incidence of severe myotoxicity

Method used

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  • Establishment of methodology for detecting genes affecting efficacy of lipid-lowering drugs by TaqMan-MGB probe technique
  • Establishment of methodology for detecting genes affecting efficacy of lipid-lowering drugs by TaqMan-MGB probe technique
  • Establishment of methodology for detecting genes affecting efficacy of lipid-lowering drugs by TaqMan-MGB probe technique

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specific Embodiment approach

[0063] The present invention will be further described in detail below with reference to the drawings and embodiments. It should be understood that the drawings and embodiments are for those skilled in the art to understand the technical solution of the present invention more easily, but not as a limitation of the protection scope of the present invention.

[0064] In the following introduction, the terms "first" and "second" are only used for the purpose of description, and should not be understood as indicating or implying relative importance. The following description provides multiple embodiments of the present invention, and different embodiments can be replaced or combined in combination, so the present invention can also be considered to include all possible combinations of the same and / or different embodiments described. Thus, if one embodiment contains features A, B, C, and another embodiment contains features B, D, then the invention should also be considered to inclu...

Embodiment 1

[0065] Example 1: Primers and TaqMan / MGB probe screening of the TaqMan / MGB probe real-time fluorescent PCR detection system that affects the therapeutic effect of lipid-lowering drugs

[0066] Two or less degenerate bases are allowed at the same variable site in the design of primers and probes. Screen the extracted candidate primers that meet the following requirements: ①The length of the probe (L) is between 19-28bp, and the length of the MGB probe is between 13-20bp; ②The Tm value is between 58-61°C; ③GC% between 25-75%; ④ poLyN≤4bp; ⑨ Perform BLAST screening, specificity score > L×0.4. The optimal Tm value is set at 61°C, and the optimal MGB probe length is 13-18bp. The labels at both ends of the probe sequence are 5'FAM-3'MGB, 5'HEX-3'MGB, 5'ROX-3'BHQ2. The screening principle of primers / probes is: select primers and probes with higher PCR amplification efficiency and probe binding efficiency in the target region as candidate primers and probes.

[0067] According to ...

Embodiment 2

[0098] Example 2: TaqMan / MGB probe real-time fluorescent PCR detection of genes affecting the efficacy of lipid-lowering drugs

[0099] 1) Extraction of human genomic DNA

[0100] For the extraction of human peripheral blood DNA, the Genomic DNA was extracted using Jiangsu Baishinuo Medical Technology Co., Ltd. Simple Extraction Whole Blood Genomic DNA Kit. The extracted sample DNA was used for subsequent experiments or stored at -20°C.

[0101] 2) Configuration of PCR reaction system

[0102] The detection system includes 6 kinds of reaction solutions: SLCO1B1 c.521T>C reaction solution, SLCO1B1 c.388A>G reaction solution, SLCO1B1 c.-910G>A reaction solution, ApoE c.388T>C reaction solution, ApoE c.526C> T reaction solution and NPC1L1 c.-18C>A reaction solution, each reaction solution contains its corresponding primer, TaqMan / MGB probe, PCR reaction buffer (Tris, betaine, potassium chloride, glycerol, Tween-20, Magnesium chloride, dNTP), TaqDNA polymerase (purchased from Fa...

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Abstract

The invention discloses a method for detecting genes affecting the efficacy of lipid-lowering drugs by combining a TaqMan-MGB probe and a real-time fluorescence quantitative PCR technique. The polymorphism of the genes affecting the efficacy of the lipid-lowering drugs in samples to be detected is detected, six SNP loci can be detected simultaneously, used primers and the TaqMan-MGB probe have high specificity, the risk of false positive caused by PCR product contamination can be reduced, the operation is simple and the result is accurate and reliable. According to the method, functional meaningful mutations such as SLCO1B1 c.521T>C (rs4149056), SLCO1B1 c.388A>G(rs2302683), SLCO1B1 c.-910G>A(rs4149015), ApoE c.388T> C(rs429358), ApoE c.526C>T(rs7412) and NPC1L1 c.-18C>A(rs41279633) are detected to assist clinicians to select appropriate drugs and doses or avoid occurring of drug interactions, the best therapeutic effect for patients is achieved, and thus the purpose of real ''individualized medication'' is achieved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a gene SLCO1B1 c.521T>C, SLCO1B1 c.388A>G, SLCO1B1 c.-910G>A, ApoE c.388T>C, ApoE c.526C that affect the curative effect of lipid-lowering drugs The detection methods of >T and NPC1L1 c.-18C>A genotypes and their special primers and probes use TaqMan / MGB probe technology to quickly perform genomic DNA amplification and fluorescent signal detection through the Real-TimePCR analysis system. Background technique [0002] According to statistics, the number of patients with hyperlipidemia in my country has reached 160 million, and 25 million people over the age of 35 suffer from both hypertension and hyperlipidemia. Although dyslipidemia has no obvious symptoms, once it occurs, it may cause disability or death. However, Chinese residents do not pay enough attention to dyslipidemia. They are not aware of the harmfulness of dyslipidemia. A large number of people with dys...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/6858C12N15/11
Inventor 刘哲张陆明姜柳
Owner 江苏百世诺医疗科技有限公司
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