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Method for distinguishing individualized medication of ramiprilat by mass spectrometry through detection products

A technology of mass spectrometry and products, which is applied in the direction of biochemical equipment and methods, microbiological determination/inspection, etc. It can solve problems such as cumbersome operation, difficulty in meeting fast and accurate requirements, and limitations of detection objects.

Inactive Publication Date: 2020-05-01
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 distinguishing individualized medication of ramiprilat by mass spectrometry through detection products
  • Method for distinguishing individualized medication of ramiprilat by mass spectrometry through detection products
  • Method for distinguishing individualized medication of ramiprilat by mass spectrometry through detection products

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Example 1: Primer Design and Synthesis

[0084] 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.

[0085] The corresponding specific PCR primer core sequences (SEQ1a to SEQ2a) and specific extension primer core sequences (SEQ1b to SEQ2b) were designed for the polymorphic sites of rs4359 and rs4344 related to the discrimination of drug types. 2 pairs of PCR primers and 2 extension primers constitute an independent reaction system: SEQ1a / b to SEQ2a / b. 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.

[0086] Related primers were synthesized at Sangon Bioengineering (Shanghai) Co., Ltd.

Embodiment 2

[0087] Embodiment 2: sample DNA extraction

[0088] 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

[0089] Embodiment three: biological experiment

[0090] Using ABI 9700 type PCR instrument, according to the instruction manual, check the 2 polymorphic sites for identifying the drug type.

[0091] The components used in the kit for PCR, PCR product purification and single base extension are:

[0092] 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

[0093] The concentration of each primer pair is 500nmol / L.

[0094] According to the manual, the specific operation method is as follows:

[0095] 1. PCR amplification

[0...

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Abstract

The invention provides a method for distinguishing individualized medication of ramiprilat by mass spectrometry through detection products, wherein the method is finally obtained by using a principlethat different single nucleotide polymorphism (SNP) loci have extension primers with different molecular weights, so that multiple loci can be simultaneously detected through mass array matrix-assisted laser desorption ionisation-time-of-flight mass spectrometry (MALDI-TOF MS). The method includes the following steps: respectively designing multiple amplification primers and extension primers according to a plurality of 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 a single-base extension reaction on the plurality of target SNP loci by using multiple sets of primers; and performing time-of-flight mass spectrometry analysis on products obtained after the single-base extension reaction, and identifying different medicine metabolism-related SNP genotypes according to products of extension primers with different molecular weights represented by mass spectrum peaks to guide individualized medication of the antihypertensive medicine ramiprilat. The method can simultaneously detect two ramiprilat medicine metabolism-related SNP loci, and has the advantages of low costs, no need to synthesize probes, short time consumption, simple and convenient result analysis, and an extremely wide range of 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 two 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 Remy Purley's personalized 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 research. T...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/6858
CPCC12Q1/6858C12Q1/6883C12Q2600/106C12Q2600/156C12Q2600/16C12Q2531/113C12Q2537/143C12Q2533/101C12Q2565/627
Inventor 马庆伟钟逾刘昕超李大为
Owner BIOYONG TECH
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