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Product for detecting polymorphism of CYP3A5 gene and detection method and application of product

A gene polymorphism and specificity technology, applied in the field of biomedicine, can solve the problems of detection sensitivity and specificity limitations, misoperation of reagents, increase of wrong detection, etc., to achieve true and reliable results, accurate and reliable results, and short time-consuming Effect

Inactive Publication Date: 2019-03-01
WUHAN YZY MEDICAL SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, multiplex PCR technology contains multiple primers and probes, and the primers and probes interfere with each other, which limits the detection sensitivity and specificity of the system.
At the same time, each component in the current genetic testing kit needs to be placed separately, and mixed on site according to the ratio when used, which is prone to problems such as operational errors or reagent contamination, which brings inconvenience to the detection and increases the probability of false detection

Method used

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  • Product for detecting polymorphism of CYP3A5 gene and detection method and application of product
  • Product for detecting polymorphism of CYP3A5 gene and detection method and application of product
  • Product for detecting polymorphism of CYP3A5 gene and detection method and application of product

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0146] Prepare the kit for detecting CYP3A5 gene polymorphism of the present invention, comprise the following steps:

[0147] A). Primer and probe screening

[0148] According to the CYP3A5 gene sequence published in the NCBI database, multiple sets of primer-probe combinations were designed, PCR systems were prepared respectively, the same wild-type and mutant templates were detected, and primer-probe combinations with better amplification sensitivity and specificity were selected.

[0149] B). Primer and probe synthesis:

[0150] The primer-probe combination screened was synthesized, specifically including: CYP3A5*3 site upstream and downstream primers SEQ ID NO: 1, SEQ ID NO: 2, specific probes SEQ ID NO: 3 and SEQ ID NO: 4. And label the FAM fluorescent group at the 5' end of SEQ ID NO:3, and mark the MGB-NFQ non-luminescence quenching group at the 3' end; at the same time, carry out LNA modification at the position containing the target allele in the middle of SEQ ID NO...

Embodiment 2

[0164] The sample to be tested was detected with the kit prepared in Example 1.

[0165] In this example, three cases of genomic DNA were collected, and the genotypes were homozygous wild, homozygous mutant, and heterozygous mutant.

[0166] 1. DNA sample dilution

[0167] Take 3 samples of genomic DNA and dilute to 0.05ng / μl.

[0168] 2. Fluorescent quantitative PCR detection of samples

[0169] Add 2 μl of the sample in step 1 to 23 μl of the CYP3A5*3 detection PCR system premix of the kit in Example 1, so that the total volume of the reaction system is 25 μl, and put it into a fluorescent quantitative PCR instrument, and set it as follows Amplification reaction after PCR reaction procedure:

[0170] 10min at 37°C;

[0171] 95°C for 5 minutes;

[0172] 95°C for 15s, 60°C for 60s, 40 cycles; collect the fluorescence signals of FAM, VIC and ROX after each cycle.

[0173] 3. Analysis of test results of samples:

[0174] CYP3A5*3 site A / A homozygous wild detection results...

Embodiment 3

[0182] The sample to be tested was detected with the kit prepared in Example 1.

[0183] In this example, two cases of genomic DNA were collected, and the genotypes were homozygous wild and homozygous mutations, respectively.

[0184] 1. DNA sample preparation

[0185] Two cases of genomic DNA samples were taken and quantified to 250ng / μl.

[0186] 2. Fluorescent quantitative PCR detection of samples

[0187] Add 2 μl of the sample in step 1 to 23 μl of the CYP3A5*3 detection reaction system of the kit in Example 1, so that the total volume of the reaction system is 25 μl, and put it into a fluorescent quantitative PCR instrument, and set up the PCR reaction as follows Amplification reaction after the program:

[0188] 10min at 37°C;

[0189] 95°C for 5 minutes;

[0190]95°C for 15s, 60°C for 60s, 40 cycles; collect the fluorescence signals of FAM, VIC and ROX after each cycle.

[0191] 3. Analysis of test results of samples:

[0192] CYP3A5*3 site A / A homozygous wild d...

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Abstract

The invention relates to the field of biomedicine, in particular to a product for detecting polymorphism of a CYP3A5 gene and a detection method and application of the product. A primer composition for detecting the polymorphism of the CYP3A5 gene is provided and comprises a primer group used for detecting CYP3A5*3 sites, wherein fluorescent reporter groups of a wild type specific probe and a mutant type specific probe are different from each other. The primer composition can accurately detect the polymorphism of the CYP3A5 gene, has the advantages of being high in sensitivity and good in specificity, can accurately detect the genotype of 0.1 ng genome DNA, and can also tolerate 500 ng genome DNA. Besides, the invention provides an enzyme-containing multiplex PCR detection kit. The detection process is greatly simplified.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a product for detecting CYP3A5 gene polymorphism, a detection method and application thereof. Background technique [0002] Hypertension is the main factor of stroke and coronary heart disease morbidity and death in the Chinese population, and the prevalence of hypertension in my country has shown an obvious upward trend in the past 50 years. Calcium ion channel blockers are commonly used antihypertensive drugs in clinical practice, but clinical practice has found that when taking such drugs, there are individual differences in the response of different patients to the drugs, and gene polymorphisms are the individual differences in drug responses. important influencing factors. [0003] There is a 6986A>G mutation in the third intron of the CYP3A5 gene (rs776746, CYP3A5*3). This SNP can lead to abnormal splicing of CYP3A5 mRNA, causing premature stop codons, and premature terminati...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q2531/113C12Q2563/107C12Q2545/101C12Q2545/113
Inventor 蔡从利李丽琼彭盼
Owner WUHAN YZY MEDICAL SCI & TECH
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