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Method for carrying out mass-spectrography distinguishing on trandolapril individualized medicine application through primer composition

A primer composition, trandolapril technology, applied in the field of oligonucleotide products, can solve problems such as difficulty in meeting fast and accurate requirements, limitations of detection objects, and cumbersome operations

Inactive Publication Date: 2020-05-29
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 sites of key enzyme genes on the homocysteine ​​metabolic pathway, and the detection objects are limited, and the operation is cumbersome, and it is difficult to meet the requirements of clinical fast and accurate needs

Method used

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  • Method for carrying out mass-spectrography distinguishing on trandolapril individualized medicine application through primer composition
  • Method for carrying out mass-spectrography distinguishing on trandolapril individualized medicine application through primer composition
  • Method for carrying out mass-spectrography distinguishing on trandolapril individualized medicine application through primer composition

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Example 1: Primer Design and Synthesis

[0087] 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.

[0088] The corresponding specific PCR primer core sequences (SEQ1a to SEQ5a) and specific extension primer core sequences (SEQ1b to SEQ5b) were designed for five polymorphic sites related to the discrimination of drug types, including rs699, rs2016848, rs11209716, rs4742610, and rs8012552. 5 pairs of PCR primers and 5 extension primers form 3 independent reaction systems: SEQ1a / b to SEQ2a / b form the first reaction system, SEQ3a / b to SEQ4a / b form the second reaction system, and SEQ5a / b forms the The third reaction system. In these 3 independent reaction systems, SEQ1a to SEQ2a, SEQ3a to SEQ4a, and SEQ5a respectively participate in 3 independent multiplex PCR reactions, and SEQ1b to...

Embodiment 2

[0090] Embodiment 2: sample DNA extraction

[0091] 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 QIAGEN’s DNeasy Blood and Tissuekit) was used to extract human genomic DNA from 200 μL of whole blood of each patient, and the DNA was extracted using a NanoDrop 2000 ( Thermo Company) quantified, and standardized to 30ng / μL (respecti...

Embodiment 3

[0092] Embodiment three: biological experiment

[0093] Using ABI 9700 type PCR instrument, according to the instructions, check the 5 polymorphic sites that are used to identify the drug type.

[0094] The components used in the kit for PCR, PCR product purification and single base extension are:

[0095] 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

[0096] The concentration of each primer pair is 500nmol / L.

[0097] According to the manual, the specific operation method is as follows:

[0098] 1. PCR amplification ...

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Abstract

The invention provides a method for carrying out mass-spectrography distinguishing on trandolapril individualized medicine application through a primer composition. The method comprises the followingsteps of: according to a plurality of objective SNP sites to be detected, independently designing a multiplex amplification primer and an extension primer; preparing a multiplex amplification primer reaction system and an extension primer reaction system; in the reaction systems, adopting multiple sets of primers to simultaneously independently carry out amplification and single-base extension reaction on the plurality of objective SNP (single nucleotide polymorphism) sites; and carrying out flight time mass spectrometry on a product subjected to the single-base extension reaction, identifyingthe genotypes of different medicine metabolism related SNPs, and guiding the individualized medicine application of a hypertension lowering medicine, i.e., the trandolapril. The method disclosed by the invention can simultaneously detect five trandolapril medicine metabolism related SNP sites, has the advantages of low cost, short time consumption, simple and convenient result analysis and wide application ranges, does not need to carry out probe synthesis, and can be used for the auxiliary diagnosis and treatment of a hypertension patient who clinically needs to take the trandolapril.

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 utilizes 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 groups. Puli'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 h...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q1/6883C12Q2600/106C12Q2600/156C12Q2600/16C12Q2531/113C12Q2537/143C12Q2533/101C12Q2565/627
Inventor 马庆伟张海燕刘昕超
Owner BIOYONG TECH
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