Method for discriminating individualized verapamil medication by mass spectrometry through primer composition

A technology of primer composition and mass spectrometry, applied in the field of oligonucleotide products, can solve the problems of unsuitability for multiple SNP detection, limitation of detection objects, rising cost, etc., to achieve high-quality medical services, simple and convenient result analysis, and low cost Effect

Inactive Publication Date: 2020-05-19
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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  • Method for discriminating individualized verapamil medication by mass spectrometry through primer composition
  • Method for discriminating individualized verapamil medication by mass spectrometry through primer composition
  • Method for discriminating individualized verapamil medication by mass spectrometry through primer composition

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] Example 1: Primer Design and Synthesis

[0090] 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.

[0091] The corresponding specific PCR primer core sequences (SEQ1a to SEQ8a) and specific extension primer core sequences ( SEQ1b to SEQ8b). 8 pairs of PCR primers and 8 extension primers (SEQ1a / b to SEQ8a / b) constitute 4 independent reaction systems. SEQ1a / b to SEQ2a / b form the first reaction system, SEQ3a / b to SEQ4a / b form the second reaction system, SEQ5a / b to SEQ6a / b form the third reaction system, and SEQ7a / b to SEQ8a / b form the The fourth reaction system. In these 4 independent reaction systems, SEQ1a to SEQ2a, SEQ3a to SEQ4a, SEQ5a to SEQ6a, SEQ7a to SEQ8a participate in 4 independent multiplex PCR reactions, SEQ1b to SEQ2b, SEQ3b to SEQ4b, SEQ5b to SEQ6b, SEQ7b to SEQ8b...

Embodiment 2

[0093] Embodiment 2: sample DNA extraction

[0094] 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

[0095] Embodiment three: biological experiment

[0096] Using ABI9700 PCR instrument, according to the instruction manual, 8 polymorphic sites for identifying the drug type were tested.

[0097] The components used in the kit for PCR, PCR product purification and single base extension are:

[0098] 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

[0099] The concentration of each primer pair is 500nmol / L.

[0100] According to the manual, the specific operation method is as follows:

[0101] 1. PCR amplification

[0102]...

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Abstract

The invention provides a method for discriminating individualized verapamil medication by mass spectrometry through a primer composition. The method comprises the following steps: respectively designing multiple amplification primers and extension primers according to eight to-be-detected target SNP loci; preparing a multiplex amplification primer reaction system and an extension primer reaction system; in the reaction systems, simultaneously and respectively carrying out amplification and single-base extension reaction on the eight target SNP loci by using a plurality of sets of primers; andcarrying out time-of-flight mass spectrometry analysis on a product after the single base extension reaction, identifying genotypes of SNP related to different drug metabolism according to products ofextension primers with different molecular weights represented by a mass spectrum peak, and guiding the medication of hypotensive drug verapamil. The method can simultaneously detect eight SNP loci related to metabolism of the hypotensive drug verapamil, and has the advantages of low cost, no need of probe synthesis, short time consumption, simple and convenient result analysis and extremely wideapplication field.

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 extensions in four multiplex PCR reactions Amplified oligonucleotide products. More specifically, the method uses different time-of-flight mass spectrometry characteristic peaks generated by different target oligonucleotide fragments in the process of mass spectrometry typing, and simultaneously detects multiple target SNP sites to guide the antihypertensive drug Vera The use of Pami. 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 research. The human genome ...

Claims

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Application Information

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