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Method for detecting telomerase activity in liquid biopsy

A technology of liquid biopsy and telomerase, which is applied in biochemical equipment and methods, and microbial measurement/inspection, etc., can solve the problems of large sample volume, difficulty in obtaining probes, and difficult promotion, and achieve high affinity and ensure efficiency. Effect

Inactive Publication Date: 2019-01-25
江苏凯强医学检验有限公司
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Problems solved by technology

[0004] The earliest detection method for telomerase activity is the telomeric repeat sequence extension method, which has low sensitivity, long detection time, and a large amount of sample required for detection, but the amount of radioactive isotopes used in the detection is high, and it is easy to pollute the environment
Commonly used detection methods for telomerase activity include telomeric repeat sequence amplification method and hybrid protection method, etc. The former method is highly sensitive and easy to operate, but it depends on the quality of the sample and too much protein will affect the experimental results: the latter method Although the method has good specificity and high sensitivity, it requires special instruments, and the required probes are difficult to obtain, so it is not easy to promote

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  • Method for detecting telomerase activity in liquid biopsy
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  • Method for detecting telomerase activity in liquid biopsy

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Embodiment Construction

[0026] Below in conjunction with embodiment the present invention is further described in detail.

[0027] A method for detecting telomerase activity in liquid biopsy of the present invention comprises the following steps:

[0028] (1) Urine sample pretreatment, and use negative screening method to remove immune cells and monocytes, add 200ul mixture (200ul 1x trap buffer, 10ng bio-TS, 1ul dNTPs, 0.5U / ulRII), the sequence of biotin-TS is 5'-biotin-AATCCGTCGAGCAGAGTT-3';

[0029] (2) Put the 1.5ml EP tube into the sonicator, and sonicate the cells;

[0030] (3) After the fragmentation is completed, put the 1.5ml EP tube into a water bath at 30 degrees Celsius and incubate for 30 minutes to allow the telomerase primer to undergo an extension reaction. Telomerase will combine with the GTT at the end of the TS primer to synthesize AGGGTTA, and then synthesize AGGGTTA after each transposition A 6-base repeat of GGTTAG;

[0031] (4) Add 10ul avidin-labeled magnetic beads to the 1...

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Abstract

The invention discloses a method for detecting telomerase activity in liquid biopsy, which utilizes avidin-labeled magnetic beads to adsorb and label the extended product with biotin after screening cancer cells, thereby achieving the effect of removing proteins and other impurities in the liquid. Compared with the prior art, the invention has the advantages that: At that end of screen cancer cell, 1*TRAP system, biotin-labeled TS primer, dNTP, RII were added. After the cells were disrupted by ultrasound, the telomerase activity was elongated by 30 DEG C water bath for 30 minutes, and then magnetic beads were added to remove the liquid. Finally, tRAP PCR reaction system was added to carry out PCR reaction with magnetic beads. This method can completely remove the influence of protein and other impurities, and the magnetic beads labeled with avidin have super affinity with the target product labeled with biotin, which can ensure the efficiency of extraction.

Description

technical field [0001] The invention relates to a detection method, in particular to a method for detecting telomerase activity in liquid biopsy. Background technique [0002] Telomeres are special structures at the ends of chromosomes in eukaryotic cells. Human telomeres are composed of 6 base repeats (TTAGGG) and binding proteins. Telomeres have important biological functions, which can stabilize the function of chromosomes, prevent chromosomal DNA degradation, end fusion, and protect chromosome structure. Due to the problem of linear DNA terminal replication in normal cells, telomeres gradually shorten as somatic cells continue to proliferate. When the cell telomere shrinks to a certain extent, the cell stops dividing and enters the aging stage. Therefore, some people call telomere the "mistosis clock" of normal cells. The length and stability of telomeres determine the lifespan of cells and are closely related to cell aging and canceration. [0003] Telomerase (Telom...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/686C12Q1/48
CPCC12Q1/48C12Q1/686C12Q2525/151C12Q2533/101C12Q2563/131C12Q2563/143C12Q2563/149
Inventor 顾凯强葛远龙李国庆杨奇飞黄洪斌周君
Owner 江苏凯强医学检验有限公司
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