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

A probe and gene technology, applied in the field of TaqMan-MGB probe technology to detect the genetics that affect the efficacy of antihypertensive drugs, can solve the problems of unavoidable clinical drug side effects, increase the financial burden of patients, and delay disease treatment, etc. Detection cycle, saving detection cost, easy to interpret the effect

Inactive Publication Date: 2019-06-18
江苏百世诺医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, clinical drug selection is administered through trial and error, that is, empirical drug use. According to statistics, the best antihypertensive drug effect is only 20-70%, and clinical drug side effects are unavoidable. This invalid and unreasonable Medication increases the economic burden of patients and delays the treatment of diseases. Therefore, formulating different treatment plans according to the genetic variation of metabolic enzymes, transporters and receptors related to individual drug treatment, realizing individualized drug treatment is not only a modern pharmacogenetic The development direction of medicine and clinical pharmacotherapeutics, and has very important social and economic significance

Method used

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

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specific Embodiment approach

[0058] 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.

[0059] 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

[0060] Example 1: Primers and TaqMan / MGB probe screening of the TaqMan / MGB probe real-time fluorescent PCR detection system for antihypertensive drug efficacy genes

[0061] 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.

[0062] Primers and TaqMan / MGB probes we...

Embodiment 2

[0088] Example 2: TaqMan / MGB probe real-time fluorescent PCR detection of genes affecting the efficacy of antihypertensive drugs

[0089] 1) Extraction of human genomic DNA

[0090] 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.

[0091] 2) Configuration of PCR reaction system

[0092] The detection system includes 5 kinds of reaction solutions: NEDD4L c.-326G>A reaction solution, AGTR1c.*86A>C reaction solution, ACE Indel reaction solution, ADRB1 c.1165G>C reaction solution, CYP3A5*3 reaction solution, each reaction The solution contains its corresponding primers, TaqMan / MGB probes, PCR reaction buffer (Tris, betaine, potassium chloride, glycerol, Tween-20, magnesium chloride, dNTP), Taq DNA polymerase (purchased from Fei Peng Biological Co., Ltd. Company, product...

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Abstract

The invention discloses a method for detecting genes affecting the efficacy of antihypertensive 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 antihypertensive drugs in samples to be detected is detected, five 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, functionalmeaningful mutations such as NEDD4L-326G>A(rs4149601), AGTR1 1186A>C(rs5186), ACE I / D(rs1799752), ADRB1 1165G>C(rs1801253) and CYP3A5*3(rs776746) 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 the detection of genotypes of NEDD4L c.-326G>A, AGTR1c.*86A>C, ACE Indel, ADRB1 c.1165G>C and CYP3A5*3 genes that affect the curative effect of antihypertensive drugs Methods and its special primers and probes, using TaqMan / MGB probe technology, rapid genomic DNA amplification and fluorescent signal detection through Real-TimePCR analysis system. Background technique [0002] Hypertension is one of the most independent and important risk factors among many risk factors for cardiovascular and cerebrovascular diseases. Cardiovascular and cerebrovascular diseases, represented by hypertension, stroke and coronary heart disease, seriously endanger human health. my country is a country with a large hypertensive population. The latest Chinese population cohort study (cohort size: 500,223) published in Internal Medicine, a sub-journal of JAMA in 2016, showed that the prevalence of...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12N15/11C12Q1/6851
Inventor 张陆明刘哲姜柳
Owner 江苏百世诺医疗科技有限公司
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