Mass spectrography method for differentiation of individualized medication of lacidipine through primer composition

A primer composition and technology of lacidipine, which are applied in biochemical equipment and methods, microbial measurement/testing, recombinant DNA technology, etc., can solve problems such as inappropriate multi-SNP detection, limitation of detection objects, and rising costs. To achieve the effect of high-quality medical services, simple and convenient result analysis, and low cost

Inactive Publication Date: 2020-06-05
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 pr

Method used

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  • Mass spectrography method for differentiation of individualized medication of lacidipine through primer composition
  • Mass spectrography method for differentiation of individualized medication of lacidipine through primer composition
  • Mass spectrography method for differentiation of individualized medication of lacidipine through primer composition

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Example 1: Primer Design and Synthesis

[0086] 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.

[0087] Design corresponding specific PCR primer core sequences (SEQ1a to SEQ6a) and specific extension primer core sequences (SEQ1b to SEQ6b) for 6 polymorphic sites related to discrimination of drug types, such as rs2238032, rs2239050, rs2239128, rs10898815, rs2429427, rs588076 . 6 pairs of PCR primers and 6 extension primers (SEQ1a / b to SEQ6a / b) constitute 2 independent reaction systems. In these two independent reaction systems, SEQ1a to SEQ3a participate in an independent multiplex PCR reaction, and SEQ1b to SEQ3b participate in a subsequent independent single base extension reaction; SEQ4a to SEQ6a participate in another independent multiplex PCR reaction, SEQ4b to SEQ6b to...

Embodiment 2

[0089] Embodiment 2: sample DNA extraction

[0090] 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

[0091] Embodiment three: biological experiment

[0092] Using ABI9700 type PCR instrument, according to the instruction manual, check the 6 polymorphic sites that are used to identify the drug type.

[0093] The components used in the kit for PCR, PCR product purification and single base extension are:

[0094] 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

[0095] The concentration of each primer pair is 500nmol / L.

[0096] According to the manual, the specific operation method is as follows:

[0097] 1. PCR amplificat...

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Abstract

The invention provides a mass spectrography method for differentiation of individualized medication of lacidipine through a primer composition. According to different single nucleotide polymorphism (SNP) sites having extension primers with different molecular weights, through matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), a plurality of sites pertinentto metabolism of a hypertension reducing medicine namely lacidipine can be detected at the same time, and finally the mass spectrography method for differentiation of individualized medication of lacidipine through the primer composition is obtained. The method comprises the steps of: according to 6 target SNP sites to be detected, separately designing multiplex amplification primers and an extension primer; compounding a multiplex amplification primer reaction system and an extension primer reaction system; in the reaction systems, performing an amplification reaction and a single-base extension reaction on the 6 target SNP sites at the same time with multiple sets of primers; and performing time-of-flight mass spectrometric analysis on products after the single-base extension reaction,identifying the genotypes of different drug metabolism relevant SNPs according to extension primer products with different molecular weights and represented by mass spectrum peaks, and guiding the medication of the hypertension reducing medicine namely the lacidipine. The method disclosed by the invention can detect 6 metabolism pertinent SNP sites of the hypertension reducing medicine namely thelacidipine at the same time, and has the advantages of being low in cost, free from probe synthesis, short in time consumption, simple and convenient in result analysis, and extremely broad in 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 two multiplex PCR reactions Amplified oligonucleotide products. More specifically, the method utilizes 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 Lassi Dipine 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): C12Q1/6883C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q1/6883C12Q2600/106C12Q2600/156C12Q2600/16C12Q2531/113C12Q2537/143C12Q2533/101C12Q2565/627
Inventor 马庆伟钟逾刘昕超张海燕
Owner BIOYONG TECH
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