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Mass-spectrography method for identifying personalized medication of nitrendipine through primer composition

A primer composition and nitrendipine technology are applied in biochemical equipment and methods, microbial measurement/testing, recombinant DNA technology, etc., and can solve problems such as unsuitability for multi-SNP detection, limitation of detection objects, and rising costs. To achieve the effect of high-quality medical services, simple and convenient result analysis, and low cost

Inactive Publication Date: 2020-06-05
BIOYONG TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the detection method and kit provided by this invention patent only target the polymorphic site of the adrenergic receptor gene, and the detection objects have limitations.
Moreover, this method mainly uses the detection method of fluorescent quantitative PCR. Since fluorescent quantitative PCR needs to design specific probes for the variation of SNP sites, the throughput of this method is low, and only one polymorphic site can be measured in one experiment. Suitable for detection of multiple SNPs
In addition, if it is necessary to obtain the information of all relevant SNPs, it is necessary to conduct multiple detection tests, which will increase the cost

Method used

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  • Mass-spectrography method for identifying personalized medication of nitrendipine through primer composition
  • Mass-spectrography method for identifying personalized medication of nitrendipine through primer composition
  • Mass-spectrography method for identifying personalized medication of nitrendipine through primer composition

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Example 1: Primer Design and Synthesis

[0080] The surrounding sequences of the target SNP of this kit are queried in the db_SNP (build 132) and Hapmap (Rel 28, Phase II + III, Aug 10) databases, and these sequences are used to design multiplex PCR primers and single base extension primers.

[0081] The corresponding specific PCR primer core sequences (SEQ1a to SEQ2a) and specific extension primer core sequences (SEQ1b to SEQ2b) were designed for rs5186, rs1137617 and other two polymorphic sites related to the discrimination of drug types. Two pairs of PCR primers and two extension primers (SEQ1a / b to SEQ2a / b) constitute an independent reaction system. In this independent reaction system, SEQ1a to SEQ2a participate in an independent multiplex PCR reaction, and SEQ1b to SEQ2b participate in a subsequent independent single base extension reaction.

[0082] Related primers were synthesized at Sangon Bioengineering (Shanghai) Co., Ltd.

Embodiment 2

[0083] Embodiment 2: sample DNA extraction

[0084] A total of 10 DNA samples were collected from ordinary Chinese people, marked as A1-A10. Among them, sample collection, DNA extraction, etc. were collected according to the requirements of the instructions, and human venous blood was collected with EDTA anticoagulant tubes. According to the instructions, the collected blood should not be stored at 2-8°C for more than one week, and at -20°C for no more than one month, and can be transported in a curling box with ice or a foam box with ice. It is recommended to use fresh blood as much as possible. Genomic DNA extraction. Since this kit does not provide human genomic DNA extraction reagents, a commercial nucleic acid extraction kit (such as DNeasy Blood and Tissuekit from QIAGEN Company) was used to extract human genomic DNA from 200 μl whole blood of each patient, and the DNA was extracted using NanoDrop 2000 ( Thermo Company) quantified and normalized to 30ng / μl (A1-A10 resp...

Embodiment 3

[0085] Embodiment three: biological experiment

[0086] Using ABI9700 type PCR instrument, according to the instruction manual, check the 2 polymorphic sites for identifying the drug type.

[0087] The components used in the kit for PCR, PCR product purification and single base extension are:

[0088] serial number component name main ingredient Specification 1 PCR Primer Mix PCR primers 24μL / tube x1 tube 2 PCR reaction solution Taq enzyme, dNTP 72μL / tube x1 tube 3 Enzyme digestion reaction solution SAP enzyme 48μL / tube x1 tube 4 Extension Primer Mix extension primer 24μL / tube x1 tube 5 Extension reaction solution Single base elongase, ddNTP 24μL / tube x1 tube 6 positive control Human Genomic DNA (30ng / μL) 10μL / tube x1 tube

[0089] The concentration of each primer pair is 500nmol / L.

[0090] According to the manual, the specific operation method is as follows:

[0091] 1. PCR amplification

[00...

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Abstract

The invention provides a detection product for mass-spectrometry identification of personalized medication of nitrendipine through a primer composition. A preparation method of the detection product includes the steps of: designing multiple-amplification primers and extension primers separately according to two to-be-detected target SNP sites, preparing a multiple amplification primer reaction system and an extension primer reaction system, performing amplification and single-base extension reactions on the two target SNP sites simultaneously through multiple sets of primers in the reaction systems, performing time-of-flight mass spectrometry analysis on products obtained after the single-base extension reactions, identifying the genotypes of different SNPs related to metabolism of the drug according to extension primer products which are represented by mass spectrum peaks and have different molecular weights, and guiding personalized medication of the antihypertensive drug nitrendipine. The two SNP sites related to metabolism of the nitrendipine drug can be detected simultaneously, and the product has the advantages of low cost, no needs for synthetic probes, short time consumption, simple and convenient result analysis and extremely wide application fields.

Description

technical field [0001] The invention belongs to the field of molecular biology detection, and relates to a method for detecting multiple PCR single-base extension products by using mass spectrometry characteristic peaks and its products. The method can simultaneously detect multiple PCR single-base extension products in a multiplex PCR reaction. Increased oligonucleotide products. More specifically, the method uses different time-of-flight mass spectrometry characteristic peaks generated by different purpose oligonucleotide fragments in the process of mass spectrometry typing, and simultaneously detects multiple target SNP sites to guide the antihypertensive drug nitrene. Dipine individualized medicine. Background technique [0002] Human genetic information is stored in the genome. The Human Genome Project, an international cooperation project, was finally completed in 2002, drawing a fine map of the human genome structure and providing a reference sequence for related res...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q1/6883C12Q2600/106C12Q2600/156C12Q2600/16C12Q2531/113C12Q2537/143C12Q2533/101C12Q2565/627
Inventor 马庆伟钟逾刘昕超
Owner BIOYONG TECH
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