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Detection product for judging individualized medication of nifedipine

A technology for nifedipine and products, which is used in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc. It can solve the problems of difficulty in considering the comprehensive factors of mutual influence of genes and few detection sites, and achieve specificity. Good sex, strong specificity, low false positive effect

Inactive Publication Date: 2020-05-26
BIOYONG TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods detect few and one-sided loci, and it is difficult to consider the comprehensive factors of gene interaction.

Method used

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  • Detection product for judging individualized medication of nifedipine
  • Detection product for judging individualized medication of nifedipine
  • Detection product for judging individualized medication of nifedipine

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 multiple PCR primers and single base extension primers.

[0087] The corresponding specific PCR primer core sequences (SEQ1a to SEQ3a) and specific extension primer core sequences (SEQ1b to SEQ3b) were designed for the three polymorphic sites related to the discrimination of drug types, such as rs2238032, rs1123617, and rs3745009. 3 pairs of PCR primers and 3 extension primers form an independent reaction system: SEQ1a / b to SEQ3a / b form a reaction body.

[0088] Related primers were synthesized at Sangon Bioengineering (Shanghai) Co., Ltd.

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 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

[0091] Embodiment three: biological experiment

[0092] Using ABI 9700 type PCR instrument, according to the instruction manual, test the 3 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 detection product for judging individualized medication of nifedipine by using an extension primer with different molecular weights at different single nucleotide polymorphism(SNP) sites, detecting a plurality of sites at the same time through matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), and finally distinguishing the individualized medication of the antihypertensive drug nifedipine. A preparation method comprises the following steps: respectively designing multiple amplification primers and extension primers according to a plurality of to-be-detected target SNP loci; preparing a multiplex amplification primer reaction system and an extension primer reaction system; in a reaction system, using a plurality of primersfor simultaneously and respectively carrying out amplification and single base extension reaction on a plurality of target SNP sites; 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 individualized medication of the antihypertensive drug nifedipine. The product can detect three SNP sites related to nifedipine metabolism at the same time, and hasthe 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 one multiplex PCR reaction Amplified oligonucleotide products. More specifically, this method uses 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, and finally distinguishes the antihypertensive drug nitrate. Individualized dosage form of bendipine. 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 seq...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/6883C12Q2600/106C12Q2600/156
Inventor 马庆伟刘昕超
Owner BIOYONG TECH
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