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Method for carrying out mass-spectrography differentiation on nitrendipine individualized medication through detection product

A technology of nitrendipine and mass spectrometry, which is applied in the field of oligonucleotide products, can solve the problems of inappropriate 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: 2020-06-05
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 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 carrying out mass-spectrography differentiation on nitrendipine individualized medication through detection product
  • Method for carrying out mass-spectrography differentiation on nitrendipine individualized medication through detection product
  • Method for carrying out mass-spectrography differentiation on nitrendipine individualized medication through detection product

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Example 1: Primer Design and Synthesis

[0078] 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.

[0079] The corresponding specific PCR primer core sequences (SEQ1a to SEQ2a) and specific extension primer core sequences (SEQ1b to SEQ2b) were designed for two polymorphic sites related to the discrimination of drug types, such as rs5186 and rs1137617. Two pairs of PCR primers and two extension primers (SEQ1a / b to SEQ2a / b) constitute an independent reaction system. In this independently performed reaction system, SEQ1a to SEQ2a participate in an independent multiplex PCR reaction, and SEQ1b to SEQ2b participate in a subsequent independent single base extension reaction.

[0080] Related primers were synthesized at Sangon Bioengineering (Shanghai) Co., Ltd.

Embodiment 2

[0081] Embodiment 2: sample DNA extraction

[0082] 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

[0083] Embodiment three: biological experiment

[0084] Using ABI9700 type PCR instrument, according to the instruction manual, check the 2 polymorphic sites for identifying the drug type.

[0085] The components used in the kit for PCR, PCR product purification and single base extension are:

[0086] 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

[0087] The concentration of each primer pair is 500nmol / L.

[0088] According to the manual, the specific operation method is as follows:

[0089] 1. PCR amplification

[00...

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Abstract

The invention discloses a method for carrying out mass-spectrography differentiation on nitrendipine individualized medication through a detection product. The method comprises the following steps of:according to two target SNP (single nucleotide polymorphism) sites to be detected, independently designing multiplex amplification primers and an extension primer; preparing a multiplex amplificationprimer reaction system and an extension primer reaction system; in the reaction systems, adopting multiple sets of primers to simultaneously independently carry out an amplification reaction and a single base extension reaction on the two target SNP sites; carrying out time-of-flight mass spectrometry analysis on products obtained by the single base extension reaction; according to extension primer products with different molecular weights and represented by mass spectrometry peaks, identifying the genotypes of different SNPs related to medicine metabolism; and guiding the individualized medication of a hypertension-reducing medicine, i.e., nitrendipine. The method can simultaneously detect the two SNP sites related to nitrendipin metabolism, has the advantages of low cost, short time consumption and an extremely wide application field and does not need to carry out probe synthesis, and result analysis is simple and convenient.

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 extension products in a multiplex PCR reaction. Increased 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. Dipine individualized medicine. 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 re...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12N15/11C12Q1/686
CPCC12Q1/686C12Q1/6883C12Q2600/106C12Q2565/627C12Q2537/143
Inventor 马庆伟刘昕超张海燕李大为
Owner BIOYONG TECH
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