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Detection product for differentiating individualized medication of Losartan

A product and medication technology, applied in the field of oligonucleotide products, can solve problems such as limitations

Inactive Publication Date: 2020-05-22
BIOYONG TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the method and kit provided by this invention patent can only detect polymorphic sites on the homocysteine ​​metabolism pathway, and cannot detect polymorphic sites not involved in homocysteine ​​metabolism , has limitations in clinical medication guidance

Method used

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  • Detection product for differentiating individualized medication of Losartan
  • Detection product for differentiating individualized medication of Losartan
  • Detection product for differentiating individualized medication of Losartan

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Example 1: Primer design and synthesis

[0083] The target SNP peripheral sequence of this kit is searched in db_SNP (build 132) and Hapmap (Rel 28, PhaseⅡ+Ⅲ, Aug 10) database, and use these sequences to design multiple PCR primers and single base extension primers.

[0084] Design the corresponding specific PCR primer core sequence (SEQ1a) and specific extension primer core sequence (SEQ1b) for the polymorphic sites of rs1057910 related to the identification of the drug type. One pair of PCR primers and one extension primer form an independent reaction system: SEQ1a / b. In this independent reaction system, SEQ1a participates in an independent multiplex PCR reaction, and SEQ1b participates in a subsequent independent single-base extension reaction.

[0085] The related primers were synthesized in Shenggong Bioengineering (Shanghai) Co., Ltd.

Embodiment 2

[0086] Example 2: Sample DNA extraction

[0087] A total of 10 samples of ordinary Chinese DNA were collected, and they were labeled A1-A10. Among them, sample collection, DNA extraction, etc. were collected from human venous blood in accordance with the requirements of the instructions, and collected with an EDTA anticoagulant tube. According to the instructions, the collected blood should be stored at 2-8°C for no more than one week, and stored at -20°C for no more than one month. It can be transported in a curling pot 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) is used to extract human genomic DNA from 200 μL of whole blood from each patient, and NanoDrop 2000 ( Thermo Company) quantified and standardized to 30ng / μL (A1-A10 respectively). Among them...

Embodiment 3

[0088] Example 3: Biological experiment

[0089] Use the ABI 9700 PCR machine to test and identify the polymorphic sites of the drug type according to the instructions.

[0090] The components used in the kit for PCR, PCR product purification and single base extension are:

[0091] Serial number Component name Main ingredients 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 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 quality control Human genomic DNA (30ng / μL) 10μL / tube x1 tube

[0092] The concentration of each primer pair is 500nmol / L.

[0093] According to the instructions, the specific operation method is as follows:

[0094] 1. PCR amplification

[0095] 1.1 In the PCR preparation area, prepare a 200μL PCR reaction tube according to the number of sample...

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Abstract

The invention provides a detection product for differentiating the individualized medication type of Losartan. A preparation method comprises the steps of according to a plurality of objective SNP sites to be detected, respectively designing a multiplex amplification primer and an extension primer; 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 on the objective SNP sites at the same time with multiple sets of primers; and performing flight time mass spectrometric analysis on products after the single-base extension reaction, identifying the genotypes of different drug metabolism relevant SNPs according to products of extension primers having different molecular weight, represented by mass spectrum peaks, and guiding the medication of the hypertension reducing medicine namely the Losartan. Through the adoption of the detection product disclosed bythe invention, SNP sites pertinent to the metabolism of the hypertension reducing medicine namely the Losartan can be detected, and the detection product has the advantages of being low in cost, freefrom 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 and product for detecting multiple PCR single-base extension products using mass spectrogram characteristic peaks. The method can simultaneously detect multiple PCR single-base extensions in two multiplex PCR reactions Amplified oligonucleotide product. More specifically, this method uses different time-of-flight mass spectrometry characteristic peaks generated by different target oligonucleotide fragments during the mass spectrometry typing process to detect multiple target SNP sites at the same time to guide the antihypertensive drug losha Tan's medication. Background technique [0002] Human genetic information is stored in the genome. In 2002, the final completion of the Human Genome Project, an international cooperation project, produced a detailed map of the human genome structure and provided reference sequences for related research. The human genome sequence...

Claims

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Application Information

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