Detection product for differentiating enalapril individualized medication types

An enalapril and product technology, applied in the field of oligonucleotide products, can solve problems such as difficulty in meeting fast and accurate needs, limitations of detection objects, and cumbersome operations

Inactive Publication Date: 2020-05-08
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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  • Detection product for differentiating enalapril individualized medication types
  • Detection product for differentiating enalapril individualized medication types
  • Detection product for differentiating enalapril individualized medication types

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0088] Example 1: Primer Design and Synthesis

[0089] 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.

[0090] 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 rs699, rs2016848, rs5522, rs11209716, rs8012552, rs495828 . 6 pairs of PCR primers and 6 extension primers form 3 independent reaction systems: SEQ1a / b to SEQ3a / b form the first reaction system, SEQ4a / b form the second reaction system, and SEQ5a / b to SEQ6a / b form the reaction system The third reaction system. In these 3 independent reaction systems, SEQ1a to SEQ3a, SEQ4a, SEQ5a to SEQ6a respectively participate in 3 independent multiplex PCR reactions, and SEQ1b to SE...

Embodiment 2

[0092] Embodiment 2: sample DNA extraction

[0093] 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

[0094] Embodiment three: biological experiment

[0095] Using ABI 9700 type PCR instrument, according to the instruction manual, check the 6 polymorphic sites for identifying the drug type.

[0096] The components used in the kit for PCR, PCR product purification and single base extension are:

[0097] 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

[0098] The concentration of each primer pair is 500nmol / L.

[0099] According to the manual, the specific operation method is as follows:

[0100] 1. PCR amplification

[0...

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Abstract

The invention provides a detection product for differentiating enalapril individualized medication types. An extension primer which has different molecular weights at different single nucleotide polymorphism (SNP) sites is utilized, so that through matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MassArray matrix-assisted laser desorption ionisation-time-of-flight massspectrometry, MALDI-TOF MS), a plurality of sites can be detected at the same time, and finally the detection product for differentiating enalapril individualized medication types is obtained. A preparation method of the detection product comprises the steps of according to a plurality of objective SNP sites to be detected, respectively designing a multiplex amplification primer and an extensionprimer; compounding a multiplex amplification primer reaction system and an extension primer reaction system; in the reaction systems, performing amplification and a single-base extension reaction onthe objective 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 thegenotypes of different drug metabolism relevant SNPs according to products of different molecular weight extension primers represented by mass spectrum peaks, and guiding the medication of the hypertension reducing medicine namely the enalapril. The detection product disclosed by the invention can detect 6 enalapril drug metabolism pertinent SNP sites at the same time, and has the advantages of being low in cost, free from synthetized probes, 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 Ena. Puli's 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 g...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12N15/11C12Q1/6858
CPCC12Q1/6858C12Q1/6883C12Q2600/106C12Q2600/156C12Q2600/16C12Q2531/113C12Q2537/143C12Q2533/101C12Q2565/627
Inventor 马庆伟钟逾刘昕超张海燕李大为
Owner BIOYONG TECH
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