Hypertension gene polymorphism fluorescent PCR (polymerase chain reaction) solubility curve detecting kit and application thereof

A technology of gene polymorphism and detection kit, which is applied in the direction of microbial determination/inspection, DNA/RNA fragments, recombinant DNA technology, etc., which can solve the problems of cumbersome operation, long detection cycle, and inability to simultaneously detect high-throughput multi-sites And other issues

Pending Publication Date: 2019-03-12
众福健康科技(杭州)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] At present, there are many technologies for gene polymorphism detection, the most commonly used are polymerase chain reaction-restriction fragment length polymorphism analysis (PCR-RFLP method), sequence-specific PCR, first-generation sequencing, next-generation sequencing and other technologies However, these technologies have their own application defects, some are cumbersome to operate, the detection cycle is long, and it is impossible to perform high-throughput multi-site simultaneous detection, which makes the current clinical application of polymorphism detection technology still unsatisfactory, and has not been able to meet clinical requirements. Testing needs
[0009] Multiplex fluorescent PCR combined with high-resolution probe method melting curve method is a new type of SNP analysis technology, which is improved on the basis of HRM (high-resolution melting curve...

Method used

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  • Hypertension gene polymorphism fluorescent PCR (polymerase chain reaction) solubility curve detecting kit and application thereof
  • Hypertension gene polymorphism fluorescent PCR (polymerase chain reaction) solubility curve detecting kit and application thereof
  • Hypertension gene polymorphism fluorescent PCR (polymerase chain reaction) solubility curve detecting kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0112] 1. Reaction solution preparation

[0113] 1) Preparation of reaction solution 1

[0114] Take a 2ml cryovial, add 0.2mol / L Tris (PH8.5) 200μl, 0.5mol / L KCL 200μl, 20mmol / L MgCL 2200 μl, 100 mmol / L dNTPs 40 μl, polymerase 100 U, 100 μM CYP3A5*3 upstream primer 5 μl, 100 μM CYP3A5*3 downstream primer 1 μl, 100 μM CYP3A5*3 fluorescent probe 2 μl, 100 μM NPPA upstream primer 5 μl, 100 μM 1 μl of NPPA downstream primer, 2 μl of 100 μM NPPA fluorescent probe. Make up the volume to 1.8ml with purified water, mix well and store in freezer for later use.

[0115] 2) Preparation of reaction solution 2

[0116] Take a 2ml cryovial, add 0.2mol / L Tris (PH8.5) 200μl, 0.5mol / L KCL 200μl, 20mmol / L MgCL 2 200 μl, 100 mmol / L dNTPs 40 μl, polymerase 100 U, 100 μM CYP2C9 upstream primer 2 μl, 100 μM CYP2C9 downstream primer 10 μl, 100 μM CYP2C9 fluorescent probe 10 μl, 100 μM AGTR1 upstream primer 2 μl, 100 μM AGTR1 downstream primer 10 μl, 10 μl of 100 μM AGTR1 fluorescent probe, 2 ...

Embodiment 2

[0146] 1. Reagent sensitivity test

[0147] 1) Experimental samples, clinical samples of each genotype, the extracted product was diluted to the minimum detection limit of the kit of 0.5ng, the test was repeated 3 times, and the final melting analysis was used to judge the negative or positive result.

[0148] 2) Experimental sample loading

[0149] Add 2 μl of sensitivity samples to reaction solution 1, reaction solution 2, and reaction solution 3 respectively, and analyze the results after repeating the test 3 times for each sample.

[0150] 3) Test results

[0151] The results of the sensitive samples of the three reaction solutions were all positive, and the repeated results were consistent.

[0152] Table 5: Test results of sensitivity samples

[0153]

[0154]

[0155] 2. Specific sample test

[0156] 1) Experimental samples, using specific samples to verify the specificity of the reagent, 2 of which are Escherichia coli, 2 of BCG, and 2 of soybean genome.

[...

Embodiment 3

[0163] Embodiment 3: detect 20 cases of clinical samples

[0164] 1) Experimental samples, clinical samples are from Chifeng City Hospital, Inner Mongolia, and 20 samples were pretreated according to the method in Example 1 through the sample diluent in the kit.

[0165] 2) Experimental sample loading

[0166] Add 2 μl of diluted clinical samples to Reaction Solution 1, Reaction Solution 2, and Reaction Solution 3, and analyze the results after repeating the test for each sample once.

[0167] 3) Test results

[0168] The results of the sensitive samples of the three reaction solutions were all positive, and the test results are as follows:

[0169] Table 7: Test results of clinical samples

[0170]

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Abstract

The invention relates to a hypertension gene polymorphism fluorescent PCR solubility curve detecting kit and application thereof, and a hypertension medication gene multiple-PCR solubility curve detecting method. The hypertension gene polymorphism fluorescent PCR solubility curve detecting kit mainly comprises a primer combination, a probe combination, and PCR reaction agents such thermally activated polymerases, PCR buffer solution and dNTPs (deoxy-ribonucleoside triphosphates) and involves polymorphism detection of 7 hypertension-related genes including CYP2D6*10, CYP2C9*3, ADRB1(1165G)C), AGTR1(1166A)C), CYP3A5*3, NPPA(T2338C) and ACE(I/D); the three reaction agents are applied to perform PCR amplification and solubility analysis to determine the related polymorphism of the 7 genes to guide clinical customized application of 5 majors types of hypertension drugs. The hypertension gene polymorphism fluorescent PCR solubility curve detecting kit can easily complete detection through 3reaction tubes, and meanwhile, save a nucleic acid extraction process, simplify operation steps and achieve a simple interpretation method.

Description

(1) Technical field [0001] The invention relates to a fluorescent PCR melting curve detection kit for gene polymorphism of hypertension, an application thereof, and a multiplex PCR melting curve detection method for detecting hypertension medication genes. (2) Background technology [0002] As a chronic non-communicable disease, hypertension is also a chronic disease with high prevalence, high disability rate and heavy disease burden in my country. According to data released by the National Health and Family Planning Commission in 2016, the prevalence of hypertension among adults aged 18 and over in my country is 25.2%. Although the awareness rate, treatment rate, and control rate of hypertension in the Chinese population have improved in recent years, they are still at a relatively low level. The global burden of disease study shows that the disability-adjusted life-year (DALY) of the Chinese population due to hypertension is as high as 37.94 million person-years, accounti...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/686C12Q1/6883C12Q2600/106C12Q2600/156C12Q2537/143C12Q2527/107
Inventor 倪晓龙李亚波李春凤欧阳云
Owner 众福健康科技(杭州)有限公司
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