FKBP5 gene methylation detection primer and kit based on pyrosequencing technology

A technology of pyrosequencing and detection kit, which is applied in the direction of recombinant DNA technology, microbial measurement/inspection, biochemical equipment and methods, etc. It can solve the problems of easy misdiagnosis and missed diagnosis, complex clinical manifestations of systemic vasculitis, and lack of organization Pathological changes and other issues, to achieve the effect of strong pertinence, low cost, and convenient detection

Inactive Publication Date: 2021-10-01
新开源晶锐广州生物医药科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The clinical manifestations of systemic vasculitis are complex, and lack laboratory diagnostic criteria and clea

Method used

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  • FKBP5 gene methylation detection primer and kit based on pyrosequencing technology
  • FKBP5 gene methylation detection primer and kit based on pyrosequencing technology
  • FKBP5 gene methylation detection primer and kit based on pyrosequencing technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Establishment of FKBP5 Gene Methylation Detection Kit Based on Pyrosequencing Technology

[0038] (1) The composition of the kit: it mainly includes the following components:

[0039] 1) FKBP5 gene-specific amplification primers FKBP5-F and FKBP5-R, the sequencing fragment to be amplified is the cg03546163 site of the FKBP5 gene; match.

[0040] The amplification primers and sequencing primers were designed according to the cg03546163 target site of the FKBP5 gene using PyroMark Assay Design 2.0 software (QIAGEN), and their sequences are as follows:

[0041] Amplification primers:

[0042] FKBP5-F: 5'-Biotin-TGAAATGTAATTTTATTGTATGTGGTTTAT-3' (the 5' end is labeled with biotin Biotin);

[0043] FKBP5-R: 5'-AAATAAATTAAACAATAATTCATCCTCAC-3'.

[0044] Sequencing primers:

[0045] FKBP5-S: 5'-TTAATTATTTATATAGAATAAGTTT-3'.

[0046] 2) Reagents for amplifying the target region of the FKBP5 gene (cg03546163 site) (Akry Biological Company, Code No. AG11104): Pro ...

Embodiment 2

[0052] Example 2 Application of FKBP5 Gene Methylation Detection Kit Based on Pyrosequencing Technology

[0053] (1) Blood cell DNA extraction (TIANamp Genomic DNA Kit, DP304)

[0054] 1) Take 200 μL of fresh or frozen human blood samples (provided by volunteers diagnosed with coronary heart disease from Guangdong Provincial People’s Hospital), and add buffer GA to make up for less than 200 μL.

[0055] 2) Add 20 μL Proteinase K solution and mix well.

[0056] 3) Add 200 μL buffer GB, mix thoroughly by inversion, place at 70°C for 10 minutes to make the solution clear, and briefly centrifuge to remove water droplets on the inner wall of the tube.

[0057] 4) Add 200 μL of absolute ethanol, shake and mix well for 15 seconds, at this time flocculent precipitation may appear, centrifuge briefly to remove water droplets on the inner wall of the tube cap.

[0058] 5) Add the solution and flocculent precipitate obtained in the previous step into an adsorption column, centrifuge at...

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Abstract

The invention belongs to the technical field of methylation detection, and particularly relates to an FKBP5 gene methylation detection primer and kit based on a pyrosequencing technology. According to the FKBP5 gene methylation detection primer based on the pyrosequencing technology, provided by the invention, a methylated CpG site which is remarkably related to the expression level of the FKBP5 gene can be obtained through amplification; and the detection primer is prepared into the detection kit for detecting the methylation of the FKBP5 gene based on the pyrosequencing technology, and the methylation of the FKBP5 gene can be simply, conveniently and quickly detected at low cost, so that the precise evaluation of the methylation level of the FKBP5 gene is realized. The kit can be used for effectively detecting the methylation of the cg03546163 site of the FKBP5 gene, so that the methylation degree of related genes of a sample to be detected is obtained, and judgment on the death risk of coronary heart disease and malignant pleural mesothelioma and the onset risk of systemic vasculitis is facilitated.

Description

technical field [0001] The invention belongs to the technical field of methylation detection, and in particular relates to a primer and a kit for detection of methylation of FKBP5 gene based on pyrosequencing technology. Background technique [0002] According to statistics, the prevalence of cardiovascular disease is on the rise, and the mortality rate of cardiovascular disease is still the first cause of death among residents. Coronary heart disease is a complex disease that is affected by both environmental and genetic factors, and changes in genetic material play an important role in the occurrence and development of coronary heart disease. Therefore, screening high-risk groups of coronary heart disease based on genetic risk factors and assisting in the implementation of individualized precision treatment will effectively reduce the morbidity and mortality of coronary heart disease, and become a new trend in the prevention and treatment of coronary heart disease in the f...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/6886C12Q1/6858C12N15/11
CPCC12Q1/6883C12Q1/6886C12Q1/6858C12Q2600/118C12Q2600/154C12Q2531/113C12Q2535/122C12Q2523/125C12Q2565/301
Inventor 钟诗龙刘玉玲汪静汤雅男吴圆圆郑淑芬
Owner 新开源晶锐广州生物医药科技有限公司
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