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Method for distinguishing individualized medication of nitrendipine and atenolol through mass spectrometry by primer composition

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

Inactive Publication Date: 2020-05-22
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 distinguishing individualized medication of nitrendipine and atenolol through mass spectrometry by primer composition
  • Method for distinguishing individualized medication of nitrendipine and atenolol through mass spectrometry by primer composition
  • Method for distinguishing individualized medication of nitrendipine and atenolol through mass spectrometry by primer composition

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Experimental program
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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 multiple PCR primers and single base extension primers.

[0091] The corresponding specific PCR primer core sequences (SEQ1a to SEQ10a) and specific extension primer core sequences (SEQ1b to SEQ10b) were designed for 10 polymorphic sites related to the discrimination of drug types such as rs5186, rs1137617, rs340874, and rs2144297. 10 pairs of PCR primers and 10 extension primers (SEQ1a / b to SEQ10a / b) constitute 3 independent reaction systems. SEQ1a / b to SEQ3a / b constitute the first reaction system, SEQ4a / b to SEQ7a / b constitute the second reaction system, and SEQ8a / b to SEQ10a / b constitute the third reaction system. In these 3 independent reaction systems, SEQ1a to SEQ3a, SEQ4a to SEQ6a, SEQ7a to SEQ10a participated in 3 independent mul...

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 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

[0095] Embodiment three: biological experiment

[0096] Using ABI9700 PCR instrument, according to the instructions, 10 polymorphic sites for identifying the type of drug 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] 1...

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Abstract

The invention provides a method for distinguishing an individualized medicinal form of nitrendipine and atenolol through mass spectrometry by a primer composition. The method includes the following steps: designing multiple amplification primers and extension primers respectively according to 10 target SNP loci to be tested; 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 10 target SNP loci by using multiple sets of primers; and performing time-of-flight mass spectrometry analysis on the product after the single base extension reaction, and according to the products of different molecular-weight extension primers represented by massspectrum peaks, identifying the genotypes of different drug metabolism-related SNPs to guide the administration of the antihypertensive drug nitrendipine and atenolol. The method can simultaneously detect 10 SNP loci related to the metabolism of the antihypertensive drug nitrendipine and atenolol, and has the advantages of low costs, no need for synthesis of probes, short time consumption, simpleand convenient analysis of results, 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 three multiplex PCR reactions Amplified 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. Lol's medication. 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...

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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