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Detection product which is prepared from specific primer composition and used for judging individualized medication of benazepril

A technique of primer composition and primer combination, which is applied in the field of molecular biology detection and can solve problems such as limitations of detection objects

Inactive Publication Date: 2020-04-28
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 polymorphic sites of key enzyme genes in the homocysteine ​​metabolic pathway, and the detection objects have limitations

Method used

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  • Detection product which is prepared from specific primer composition and used for judging individualized medication of benazepril
  • Detection product which is prepared from specific primer composition and used for judging individualized medication of benazepril
  • Detection product which is prepared from specific primer composition and used for judging individualized medication of benazepril

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] Example 1: Primer Design and Synthesis

[0092] 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.

[0093] The corresponding specific PCR primer core sequences (SEQ1a to SEQ9a) and specific extensions were designed for nine polymorphic sites related to the identification of individualized drug types, including rs1042713, rs11209716, rs1799998, rs1801133, rs2016848, rs2229437, rs699, rs7079, and rs8012552 Primer core sequences (SEQ1b to SEQ9b). 9 pairs of PCR primers and 9 extension primers form 3 independent reaction systems: SEQ1a / b to SEQ3a / b form the first reaction system, SEQ4a / b to SEQ6a / b form the second reaction system, and SEQ7a / b to SEQ7a / b SEQ9a / b constitutes the third reaction system. In these 3 independent reaction systems, SEQ1a to SEQ3a, SEQ4a to SEQ6a, SEQ7a to S...

Embodiment 2

[0095] Embodiment 2: sample DNA extraction

[0096] 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

[0097] Embodiment three: biological experiment

[0098] Using ABI 9700 PCR instrument, according to the instructions, the 9 polymorphic sites that are used to identify the individualized drug type were tested.

[0099] The components used in the kit for PCR, PCR product purification and single base extension are:

[0100] 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

[0101] The concentration of each primer pair is 500nmol / L.

[0102] According to the manual, the specific operation method is as follows:

[0103] 1. PCR...

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Abstract

The invention provides a detection product for judging individualized medication of benazepril. 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 sites; preparing a multiplex amplification primer reaction system and an extension primer reaction system; in a reaction system, simultaneously and respectively carrying out amplification and single base extension reaction on the plurality of target SNP sites by using a plurality of primers; 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 mass spectrum peaks, and guiding individualized medication of antihypertensive drug benazepril. The detection product can simultaneously detect 9 SNP sites related to benazepril drug metabolism, and has the 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 two multiplex PCR reactions Amplified oligonucleotide products. More specifically, this 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 Bena 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. ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/6883C12Q2600/106C12Q2600/156
Inventor 马庆伟钟逾
Owner BIOYONG TECH
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