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Method for identifying individualized administration of verapamil by performing mass spectrometry through detection products

A mass spectrometry and product technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc. Simple and convenient medical service, result analysis, 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 the 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 multiple SNP detection
In addition, if it is necessary to obtain all relevant SNP information, it is necessary to conduct multiple detection tests, which will increase the cost

Method used

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  • Method for identifying individualized administration of verapamil by performing mass spectrometry through detection products
  • Method for identifying individualized administration of verapamil by performing mass spectrometry through detection products
  • Method for identifying individualized administration of verapamil by performing mass spectrometry through detection products

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0088] Example 1: Primer Design and Synthesis

[0089] 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.

[0090] The corresponding specific PCR primer core sequences (SEQ1a to SEQ8a) and specific extension primer core sequences ( SEQ1b to SEQ8b). Eight pairs of PCR primers and eight extension primers (SEQ1a / b to SEQ8a / b) constitute four 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, SEQ...

Embodiment 2

[0092] Embodiment 2: sample DNA extraction

[0093] 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 in accordance with 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-A1...

Embodiment 3

[0094] Embodiment three: biological experiment

[0095] Using ABI9700 PCR instrument, according to the instruction manual, 8 polymorphic sites for identifying the drug type were tested.

[0096] The components used in the kit for PCR, PCR product purification and single base extension are:

[0097] 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

[0098] The concentration of each primer pair is 500nmol / L.

[0099] According to the manual, the specific operation method is as follows:

[0100] 1. PCR amplification

[0101]...

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Abstract

The invention provides a method for identifying individualized administration of verapamil by performing mass spectrometry through detection products. The method comprises the following steps: designing multiple amplification primers and extension primers according to eight target SNP loci to be detected; preparing a multiple amplification primer reaction system and an extension primer reaction system; in the reaction systems, simultaneously performing amplification and single-base extension reactions on the eight target SNP loci by using multiple sets of primers; and performing time-of-flightmass spectrometry analysis on the products after the single-base extension reactions, and identifying the genotypes of different drug metabolism-related SNP loci according to the products of different-molecular-weight extension primers represented by mass spectrum peaks to guide the administration of the antihypertensive drug verapamil. The method can simultaneously detect the eight SNP loci related to the metabolism of the antihypertensive drug verapamil, and has the advantages of low costs, no need for synthesis of 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 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. In 2002, the Human Genome Project, an international cooperation project, was finally completed, 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): C12Q1/6883C12N15/11
CPCC12Q1/6883C12Q2600/106C12Q2600/156C12Q2600/16
Inventor 马庆伟钟逾张海燕刘昕超
Owner BIOYONG TECH
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