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A kit for detecting gene polymorphisms of hypertension drugs by multiplex fluorescent PCR method

A gene polymorphism and multiple fluorescence technology, applied in the field of fluorescent quantitative PCR, can solve the problems of low sensitivity, complicated operation, cross-contamination and other problems of sequencing methods, and achieve the effect of improving treatment efficiency, reducing drug side effects, and reducing detection costs

Active Publication Date: 2020-03-27
DEBIQI BIOTECH XIAMEN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The sensitivity of the sequencing method is low, and the entire detection cycle takes 7 days, the operation is complicated and not suitable for clinical promotion; the sensitivity of the gene chip method can only reach 10 4 copies / mL, and the operation process is likely to cause cross-contamination and lead to false positives

Method used

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  • A kit for detecting gene polymorphisms of hypertension drugs by multiplex fluorescent PCR method
  • A kit for detecting gene polymorphisms of hypertension drugs by multiplex fluorescent PCR method
  • A kit for detecting gene polymorphisms of hypertension drugs by multiplex fluorescent PCR method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] 1. Preparation of reaction solution

[0038] (1) Preparation of reaction solution 1#

[0039] Take a 10mL volumetric flask, add 0.5mol / L Trizma ® HCl 64μL, 0.5mol / L Trizma ® Base336μL, 1mol / L MgCl 2 20 μL, 1mol / L KCl 600 μL, 0.5mol / L EDTA·2Na 10 μL, DMSO 200 μL, dNTPs 90 μL, 50 μmol / L CYP2C9 upstream and downstream primers 83.4 μL each, 50 μmol / L CYP2C9 (*1) probe, CYP2C9 (*3 ) probes, 12.5 μL each, 50 μmol / L CYP2D6 upstream and downstream primers, 83.4 μL, 50 μmol / L CYP2D6 (*1) probes, and CYP2D6 (*10) probes, 12.5 μL each. Make up the volume to 10mL with double distilled water, turn it over to mix well, transfer the liquid to a 10mL beaker, divide into centrifuge tubes at 1mL per branch, and store at -20°C for later use.

[0040] (2) Preparation of reaction solution 2#

[0041]Take a 10mL volumetric flask, add 0.5mol / L Trizma ® HCl 64μL, 0.5mol / L Trizma ® Base336μL, 1mol / L MgCl 2 20 μL, 1mol / L KCl 600 μL, 0.5mol / L EDTA·2Na 10 μL, DMSO 200 μL, dNTPs 90 μL, ...

Embodiment 2

[0071] 1. Reagent specificity verification

[0072] (1) Experimental samples

[0073] Six specific samples were taken to verify the specificity of the reagent, among which 2 were normal saline samples, 2 were Escherichia coli, and 2 were calf serum samples.

[0074] (2) Experimental process

[0075] Use reaction solution 1#, reaction solution 2#, reaction solution 3#, and reaction solution 4# to detect the above 6 specific samples, analyze the test results, and verify the specificity of the reagents.

[0076] (3) Experimental results

[0077] The test results of 6 specific samples were all negative, indicating that the reagent has good specificity and no cross-reaction. The specific results are shown in the table below:

[0078] Reaction solution 1# specificity test result

[0079]

[0080] Reaction solution 2# specificity test result

[0081]

[0082] Reaction solution 3# specificity test result

[0083]

[0084] Reaction solution 4# specificity test result

...

Embodiment 3

[0116] Embodiment 3: Detect the result of 40 routine clinical samples

[0117] 1. According to the preparation method shown in Example 1, the relevant components of the kit were prepared and stored at -20°C for later use.

[0118] 2. Obtain 40 cases of clinical whole blood samples from Zhongshan Hospital Affiliated to Xiamen University, use the nucleic acid extraction reagent (whole blood type) produced by Dobe Biotechnology (Xiamen) Co., Ltd. to extract the genomic DNA of 40 cases of clinical samples, and use a UV spectrophotometer The purity of the DNA samples was tested, and the OD260 / OD280 of 40 samples were all between 1.6 and 2.0.

[0119] 3. According to the steps shown in Example 1, add DNA samples and perform detection on a fluorescent quantitative PCR instrument. The instrument used this time is ABI7500.

[0120] 4. According to the judgment standard shown in Example 1, the results are interpreted and counted, and the results are as follows:

[0121]

[0122] ...

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Abstract

The invention provides a kit for detecting gene polymorphism of a drug for hypertension by a multiple fluorescent PCR method. The kit comprises 7 types of gene polymorphism detection (CYP2C9*1 and *3, CYP2D6*1 and *10, CYP3A5*1 and *3, ADRB1(1165G > C), AGTR1(1166A > C), ACE (I / D) and NPPA (2238 T > C)) related to drug susceptibility of hypertension. Amplification is performed through four reaction buffer liquids, and each of the reaction buffer liquids comprises four fluorescent channels. The types of alleles of 7 sites are judged through an amplification result, so that clinical medication is guided.

Description

technical field [0001] The invention belongs to the field of fluorescence quantitative PCR, and in particular relates to a kit for detecting gene polymorphism of hypertension medicine by multiple fluorescence PCR method. Background technique [0002] It is conservatively estimated that the number of hypertensive patients in my country exceeds 160 million. The incidence of hypertension is on the rise, and stroke, a complication of hypertension, is the second leading cause of death in my country. Hypertension and its complications have become the third largest economic burden of disease in the world. Controlling blood pressure through drug therapy is the main means to reduce the occurrence of hypertensive complications at present and for a long time in the future. [0003] Among the top 200 best-selling medicines in the world in 2000, medicines for treating high blood pressure accounted for 17 kinds. However, clinical individual response differences in drugs for the treatme...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6858C12Q1/6883
CPCC12Q1/6883C12Q2600/156
Inventor 童超邱一帆
Owner DEBIQI BIOTECH XIAMEN
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