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Method for discriminating individualized medication of nitrendipine and atenolol through mass spectrometry by detecting product

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: 2020-05-22
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 discriminating individualized medication of nitrendipine and atenolol through mass spectrometry by detecting product
  • Method for discriminating individualized medication of nitrendipine and atenolol through mass spectrometry by detecting product
  • Method for discriminating individualized medication of nitrendipine and atenolol through mass spectrometry by detecting product

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 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 discriminating the individualized medication type of nitrendipine and atenolol through mass spectrometry by detecting a product. The method comprises the followingsteps: respectively designing multiple amplification primers and extension primers according to 10 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 10 target SNP loci by using a plurality ofsets of primers; and carrying 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 of extension primers with different molecular weights represented by a mass spectrum peak, and guiding the medication of antihypertensive drug nitrendipine and atenolol. The methodcan simultaneously detect the 10 SNP loci related to metabolism of the antihypertensive drug nitrendipine and atenolol, and has the advantages of low cost, no need of probe synthesis, short time consumption, simple and convenient result analysis and extremely wide application 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 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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IPC IPC(8): C12Q1/6883C12Q1/6872C12N15/11
CPCC12Q1/6872C12Q1/6883C12Q2600/106C12Q2600/156C12Q2537/143C12Q2531/113
Inventor 马庆伟张海燕刘昕超
Owner BIOYONG TECH
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