Telomerase activity detection method based on electrochemical luminescence cascade amplification principle

A technology of activity detection and cascading amplification, which is applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of low detection efficiency, high analysis cost, complicated operation, etc.

Active Publication Date: 2017-12-19
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing detection methods for telomerase activity have problems such as primer immobilization,

Method used

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  • Telomerase activity detection method based on electrochemical luminescence cascade amplification principle
  • Telomerase activity detection method based on electrochemical luminescence cascade amplification principle
  • Telomerase activity detection method based on electrochemical luminescence cascade amplification principle

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0100] Example 1 Technical process of telomerase activity detection method based on electrochemiluminescence cascade amplification principle

[0101] (1) Cell and tissue telomerase extraction

[0102] ① Collect 25,000 to 100,000 cells in a 1.5mL microcentrifuge tube without DNase and RNase.

[0103] ② Centrifuge the sample obtained in step ① at 3,000×g at room temperature (18-25°C) for 5 minutes in a bench top centrifuge to prepare pelleted cells. Discard the supernatant.

[0104] ③Resuspend the cell pellet obtained in step ② in ice-cold NP-40 lysis buffer at a concentration of 500-1,250 cells μL -1 , And incubate on ice for 30 minutes to obtain a whole cell lysate. Be careful not to contaminate any steps with RNase.

[0105] ④Step ③The obtained whole cell lysate can be quickly frozen in liquid nitrogen and placed at -80°C until ready for analysis.

[0106] ⑤ The whole cell lysate obtained in step ③ is centrifuged at 16,000×g for 20 minutes at 4°C in a benchtop microcentrifuge to remov...

Embodiment 2

[0130] Example 2 The feasibility of telomerase extension and electrochemiluminescence system cascade amplification system

[0131] The present invention first verifies the feasibility of the telomerase extension system. As the starting chain that triggers the extension of telomerase, TS primer is the key point of the present invention. We use biotin at its chain end to extend telomerase while providing a streptavidin capture site. The control group (not Extending telomerase) is denoted as C. The results of the telomerase extension experiment are as follows image 3 As shown in A, when telomerase is present, the electrophoretic bands appear as ladder-like extension fragments, which proves the feasibility of the telomerase extension scheme.

[0132] At the same time, we conducted a feasibility analysis on the assembly of the electrochemiluminescence cascade amplification system, and carried out the electrochemiluminescence intensity of the telomerase activity detection according to ...

Embodiment 3

[0133] Example 3 Telomerase activity detection performance, specificity evaluation and tumor cell sample detection experiment

[0134] Sensitivity and specificity are important performance indicators for measuring the new detection method. The present invention evaluates the sensitivity and specificity of telomerase activity detection in the cascade amplification system of the electrochemiluminescence system (experimental results such as Figure 4 Shown in A and B). Experimental results ( Figure 4 A) It shows that the detection sensitivity of the present invention reaches 100 cells, and a good sensitivity is achieved. At the same time, the inventor evaluated its specificity (experimental results such as Figure 4 B), which proves that the detection platform has good specificity. Only the experimental group containing telomerase gave a strong response signal, while the luminous intensity of the other experimental groups was the same as that of the control group. This proves th...

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Abstract

The invention discloses a telomerase activity detection method based on an electrochemical luminescence cascade amplification principle, which belongs to the technical field of telomerase activity detection. The invention provides the high-sensitivity telomerase activity detection method based on the electrochemical luminescence cascade amplification principle by utilizing performance that telomerase can execute adding a repetitive sequence and combining an electrochemical luminescence amplifying signal group linearity and arborescence terpyridyl ruthenium polymer electrochemical luminescence method. The method provided by the invention has the following advantages of (1) high sensitivity; (2) probe stability: wherein a linear and arborescence terpyridyl ruthenium polymer is used as an electrochemical luminescence amplification part of a probe, the performance is stable, and the polymerization degree and the luminescent intensity are uniform; (3) simple and fast detection process: wherein telomerase activity monitoring can be carried out through a simple telomerase extracting process, the time consumption is short, an amplification step is saved, and the detection is carried out quickly; (4) low cost.

Description

Technical field [0001] The invention belongs to the technical field of telomerase activity detection, and particularly relates to a telomerase activity detection method based on the principle of electrochemiluminescence cascade amplification. Background technique [0002] Telomerase is a ribonucleoprotein reverse transcriptase that uses its own RNA as a template to perform the function of adding the telomeric repeat sequence TTAGGG to the end of the chromosome. Recent studies have shown that the activation of telomerase can lead to the maintenance of telomere length and the enhancement of chromosome replication, which is closely related to the occurrence and development of human aging diseases and tumors. Therefore, the detection of telomerase activity can provide an important basis for the diagnosis, prediction and clinical treatment of cancer and aging diseases. The traditional detection method of telomerase activity (telomere repeat sequence amplification method based on poly...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/48
CPCC12Q1/6806C12Q1/682C12Q2563/103C12Q2563/143C12Q2563/149C12Q2523/301
Inventor 黄曦廖玉辉赵钊艳谭青琴
Owner SUN YAT SEN UNIV
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