Detection method of telomerase activity based on the principle of electrochemiluminescence cascade amplification
A technology of activity detection and cascade amplification, which is applied in the fields of biochemical equipment and methods, and the determination/inspection of microorganisms, which can solve the problems of low detection efficiency, complicated operation and low sensitivity.
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Embodiment 1
[0100] Example 1 Technical process of the detection method of telomerase activity based on the principle of electrochemiluminescence cascade amplification
[0101] (1) Cell and tissue telomerase extraction
[0102] ① Collect 25,000-100,000 cells in a 1.5mL microcentrifuge tube free of DNase and RNase.
[0103] ② Centrifuge the sample obtained in step ① at 3,000×g for 5 minutes at room temperature (18-25° C.) in a desktop 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 μL -1 , and incubated on ice for 30 min to obtain whole cell lysates. Take care not to contaminate any steps with RNase.
[0105] ④ Step ③ The resulting whole cell lysate can be snap-frozen in liquid nitrogen and placed at -80°C until ready for analysis.
[0106] ⑤ Centrifuge the whole cell lysate obtained in step ③ at 16,000×g for 20 minutes at 4°C in a benchtop microcentrifug...
Embodiment 2
[0130] Example 2 Feasibility of telomerase extension and electrochemiluminescence system cascade amplification system
[0131] In the present invention, the feasibility of the telomerase extension system is firstly verified. As the initial strand that triggers telomerase extension, the TS primer is the key point of the present invention. We will label its chain end with biotin 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 assay were as follows: image 3 As shown in A, when telomerase exists, the electrophoresis band appears a ladder-like extension fragment, thus proving 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. According to the technical route of telomerase activity detection in Example (1), the electrochem...
Embodiment 3
[0133] Example 3 Telomerase activity detection performance, specificity evaluation and tumor cell sample detection experiment
[0134] Sensitivity and specificity are important performance indexes to weigh the novel detection method. The present invention evaluates the detection sensitivity and specificity of electrochemiluminescent system cascade amplification system telomerase activity (experimental results are as follows: Figure 4 A, B shown). Experimental results ( Figure 4 A) shows that the detection sensitivity of the present invention has reached 100 cells, and better sensitivity has been achieved. At the same time, the inventor evaluated its specificity (experimental results such as Figure 4 Shown in B), which proves that the detection platform has good specificity, only the experimental group containing telomerase gives a strong response signal, while the luminescence intensity of other experimental groups is equal to that of the control group. Thus, it is prove...
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Abstract
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