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Method for detecting hotspot mutation of human TERT gene promoter

A technology of TERT-124 and TERT-146, applied in the field of real-time fluorescent quantitative PCR detection, can solve the problems of high detection cost, expensive detection equipment, complicated operation, etc., achieving high universality, clear and objective interpretation of results, and high detection sensitivity. Effect

Inactive Publication Date: 2020-07-17
AEROSPACE CENT HOSPITAL
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Problems solved by technology

At present, for the detection of the hotspot mutation rate of TERT, there are NGS method (the lower limit of detection is about 0.1%) and ddPCR method (the lower limit of detection is about 0.1%-0.05%) reported in the open literature. Problems such as high cost and expensive testing equipment are not suitable for clinical routine development

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  • Method for detecting hotspot mutation of human TERT gene promoter
  • Method for detecting hotspot mutation of human TERT gene promoter
  • Method for detecting hotspot mutation of human TERT gene promoter

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Embodiment Construction

[0030] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0031] The instruments used in the present invention are as follows: ABI7500 fluorescence quantitative PCR instrument, ABI2700 PCR instrument, AlphaInnotech gel imager, Bio-Rad company PowerPac electrophoresis instrument, UV spectrophotometer, high-speed centrifuge, water bath, vortex shaker, refrigerator , oven, autoclave, sterile water for injection.

[0032] Reagents used in the present invention: The following reagents are all from Tiangen Biochemical Technology (Beijing) Co., Ltd., including 2×GC-Rich premix reagent, common DNA product purification reagent, and SuperReal fluorescence quantitative premix reagent (probe method).

[0033] The primers, TaqMan probes and LNA inhibitory probes used in the present invention are all synthesized by Beijing Saibaisheng Company; TaqMan-MGB probes are synthesized by Shanghai Synthesis Department of ABI Company. ...

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Abstract

The invention discloses a method for detecting hot spot mutation of a human TERT gene promoter. The method comprises the following steps: a, enriching templates by adopting DNA enrichment conventionalPCR aiming at different specimens; b, purifying the PCR product, determining the concentration by using an ultraviolet spectrophotometer, and performing concentration calibration and standard substance preparation; and c, carrying out TERT-146 (C / T) and TERT-124 (C / T) mutation rate quantitative detection on the calibrated specimens by adopting qPCR. According to the method, a qPCR technology platform which is conventionally developed clinically is adopted, the high-sensitivity TERT hot spot mutation rate quantitative detection method is established for DNA specimens from various sources, thelower limit of the detected mutation rate reaches 0.05%, and the method has the advantages of being high in detection sensitivity, high in specificity, capable of accurately and quantitatively detecting the mutation rate, low in cost, suitable for conventional clinical development and the like.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a real-time fluorescence quantitative PCR (qPCR) detection method for detecting the mutation rates of human TERT gene promoter hot spot mutations -146 (C / T) and -124 (C / T). Background technique [0002] Telomere refers to a unique structure composed of DNA and related proteins containing simple repeating 5'-TTAGGG-3' base series at the end of chromosomes. It exists in eukaryotic cells and its main function is to maintain the integrity of chromosome structure and genome. Stability. With each cell division cycle, telomere DNA is reduced, and telomere length is progressively shortened, eventually leading to the cessation of cell division, forcing cells to senesce and die. Telomerase is a ribonucleoprotein polymerase mainly composed of telomerase RNA template (hTR), telomerase reverse transcriptase (TERT) and telomerase-related proteins. The activation of telomerase is the main mechanis...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/6851
CPCC12Q1/6886C12Q1/6851C12Q2600/118C12Q2600/156
Inventor 梁国威张洁
Owner AEROSPACE CENT HOSPITAL
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