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Kit for detecting gene polymorphism of human hypertension risk and preparation method and application thereof

A detection kit and gene technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of low sensitivity of PCR-sequencing method, low detection sensitivity, special equipment requirements, etc.

Active Publication Date: 2018-12-11
WUHAN HEALTHCHART BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The PCR-sequencing method has low sensitivity and takes a long time for the experiment, so it is not suitable for clinical promotion; the chip hybridization method has low detection sensitivity and poor specificity, and is prone to false positive results; the high-resolution melting curve method has special requirements for equipment and is not suitable for clinical promotion. Detection sensitivity is not high
The Taqman-qPCR method has high detection sensitivity, but often due to the site sequence, the detection specificity is not high, and it is genotyped by the Genotyping method, which has certain requirements for the number of samples and polymorphism distribution, and is not suitable for detecting mutation frequency too low position

Method used

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  • Kit for detecting gene polymorphism of human hypertension risk and preparation method and application thereof
  • Kit for detecting gene polymorphism of human hypertension risk and preparation method and application thereof
  • Kit for detecting gene polymorphism of human hypertension risk and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] Example 1 Preparation of Hypertension Risk Gene Detection Kit of the present invention

[0090] 1. Primer design and synthesis:

[0091] The 5 sites of CYP2C9, CYP2D6, ADRB1, AGTR1 and ACE contain 6 primers in each system; two sense chain upstream primers F1 and F2, two antisense chain downstream primers R1 and R2, F1 and R1 are common primers , F2 and R2 are specific ARMs primers with fluorophore and tag sequence, screened by primers and PCR reaction conditions to ensure that the primer pair F1R1 enriches the template, while F2 and R1, F1 and R2 can normally amplify the band Fluorescent specific genotype PCR products; at the same time, internal reference primers were added to each gene locus.

[0092] The specific sequence is as follows:

[0093] CYP2C9-F1: 5'-TTGTGGACTTCCTCTTCCTTC-3' SEQ ID NO.1

[0094] CYP2C9-F2: 5'-VIC-ATCCCTTGTCATCGTCAGAGATACA-3' SEQ ID NO.2

[0095] (Specific recognition of wild template)

[0096] CYP2C9-R1: 5'-GTCACCCCTGCCAGAAAT-3' SEQ ID N...

Embodiment 2

[0159] The human hypertension risk gene polymorphism detection kit prepared in Example 1 was used to detect the samples to be tested. In this example, peripheral blood samples from 100 healthy people were collected, genomic DNA was extracted, and human hypertension risk gene polymorphism detection kits were used to detect hypertension risk gene polymorphisms. The specific operation process was as follows:

[0160] (1) Genomic DNA extraction from blood samples: Use a commercial extraction kit to extract genomic DNA. After the extraction is complete, use TE buffer to elute the DNA and measure the DNA concentration; dilute the genomic DNA to 20ng / μl;

[0161] (2) Fluorescence quantitative detection: Add 30 μl of PCR premixed reaction solution and 20 μl of sample DNA to be tested into the reaction tube; the PCR reaction program is: 95°C for 1 minute pre-denaturation; 15 cycles: 95°C for 5 seconds, 61°C for 32 seconds , do not collect fluorescence; 30 cycles: 95°C for 5 seconds, 61...

Embodiment 3

[0185] Use the kit prepared in Example 1 to detect and verify the minimum detection line of this kit, specifically including the following steps:

[0186] (1) Using the samples of known CYP2C9, CYP2D6, ADRB1, AGTR1 and ACE corresponding site genotypes in Example 2, select one case of wild-type genome, mutant genome and heterozygous genome samples for each site, and set Dilute the above samples to 10ng / μL, 1ng / μL, 0.5ng / μL, 0.25ng / μL, 0.1ng / μL, 0.05ng / μL, 0.025ng / μL, 0.001ng / μL;

[0187] (2) Fluorescence quantitative detection: Add 30 μl of PCR premixed reaction solution and 20 μl of sample DNA to be tested into the reaction tube; the PCR reaction program is: 95°C for 1 minute pre-denaturation; 15 cycles: 95°C for 5 seconds, 61°C for 32 seconds , do not collect fluorescence; 30 cycles: 95°C for 5 seconds, 61°C for 32 seconds, collect fluorescence; each concentration gradient of each sample is repeated 20 times;

[0188] (3) After the PCR reaction is completed, perform result i...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to a kit for detecting gene polymorphism of human hypertension risk and a preparation method and application thereof.The kit respectively consists of a PCR (polymerase chain reaction) pre-mixing reaction solution, a positive reference substance and a negative reference substance, wherein the PCR pre-mixing reactionsolution is used for amplifying the polymorphism of five genes CYP2C9, CYP2D6, ADRB1, AGTR1 and ACE related with the human hypertension risk; the PCR pre-mixing reaction solution comprises a specificprimer sequence group, a probe group and a PCR reaction solution, and the specific primer sequence group is used for amplifying each site and consists of a common outer primer and a specific ARMs (amplification refractory mutation system) primer with a fluorescent label. The kit for detecting the gene polymorphism of human hypertension risk has the advantages that the sensitivity is high, the specificity is high, the operation is simple and convenient, the result is reliable, and the like; the detection can be completed within one hour, and the result judgment is simple and objective.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a human hypertension risk gene polymorphism detection kit and its preparation method and application. Background technique [0002] The number of hypertensive patients in my country is as high as hundreds of millions, and the incidence rate is still on the rise. The complications caused by it have become the second leading cause of death in my country. Controlling blood pressure through drug therapy to reduce hypertensive complications has become the main treatment for hypertension. However, it is very common that there are individual differences in clinical responses to drugs for the treatment of hypertension, and 20-50% of patients receiving drug therapy have not well controlled blood pressure. Advances in pharmacogenetics and pharmacogenomics have shown that genetic variation in drug-metabolizing enzymes, transporters, and receptors (drug targets) is a major cause of ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/686
CPCC12Q1/686C12Q1/6883C12Q2600/156C12Q2535/137C12Q2537/143
Inventor 晏星李倩李雪梅叶伦程弘夏陈刚
Owner WUHAN HEALTHCHART BIOLOGICAL TECH
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