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Telomerase activity detection kit and detection method

A detection kit and activity detection technology, which is applied in biochemical equipment and methods, measuring devices, microbe determination/inspection, etc., can solve the problems of chip damage, single use, and low efficiency of molecular modification, and achieve high specificity, The process is simple and the effect of reducing molecular mismatch problems

Active Publication Date: 2022-05-13
NANJING UNIV OF POSTS & TELECOMM
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  • Abstract
  • Description
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Problems solved by technology

[0008] For the highly sensitive detection of telomerase activity, our research group has conducted systematic research using nanopore sensors. First, we have used the principle of base complementarity to design molecular probes for telomere extension sequences. For example, the application numbers applied by our research group are 202210071448.7, The patent titled "A Detection Probe, Kit, and Direct Detection Method for Telomerase Activity" discloses a gold nanoparticle assembly that is linked by DNA, and the telomerase extension reaction causes the nanoparticle assembly to decompose. This method has high sensitivity, and at the same time solves the problems of low molecular modification efficiency, chip damage, and single use in traditional nanopore sensing. Pore ​​sensing as a new approach for lab-on-chip structure and activity-function detection of proteases

Method used

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  • Telomerase activity detection kit and detection method
  • Telomerase activity detection kit and detection method
  • Telomerase activity detection kit and detection method

Examples

Experimental program
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Embodiment 1

[0054] Example 1: Synthesis of gold nanorods

[0055] First is the synthetic seed: take 10mL, 0.1M CTAB solution and 0.25mL, 0.01M HAuCl 4 Mix, add magneton, then add 0.6 mL of freshly prepared 0.01 mol / L NaBH 4 solution, stirring vigorously for 2 minutes while adding, 30 ° C water bath for 2 hours;

[0056] Followed by the growth of gold nanorods: take 40mL 0.1M CTAB solution, 2mL 0.01M HAuCl 4 solution and 350uL 0.01M AgNO 3Mix the solution and stir with a magnet, add 800uL of 1M HCl solution to adjust the pH to about 2, add 200uL of 0.1M ascorbic acid solution, stir until the solution becomes colorless, add 200uL of the prepared gold seed solution, stir and place Grow in a water bath at 30°C for 8 hours. Centrifuge the gold nanorod solution grown for 8 hours in a 50mL centrifuge tube at 8000rpm for 30min, absorb the supernatant to retain the precipitate, add ultrapure water to redissolve, repeat three times, concentrate the centrifuged gold nanorod solution and use a wi...

Embodiment 2

[0059] Embodiment 2: the method for detecting telomerase activity

[0060] Step 1, extraction of telomerase: Add 4mL of culture medium to the cell culture dish, take out about 300uL of the cell solution in the centrifuge tube, add it to the culture dish, shake gently to make the cells evenly distributed, and put it into the incubator to grow to maintain HeLa, MCF-7 and LO2 cells. Various cells were collected during the exponential growth phase and washed twice with ice-cold PBS buffer (0.01M, pH 7.4), and then the cells were resuspended in 200 μL of ice-cold CHAPS lysis buffer (10 mM pH 7.5 Tris-HCl, 1 mM MgCl2, 1 mMEGTA, 0.5% CHAPS, 10% glycerol, 0.1 mM PMSF). The lysate was incubated in an ice-water bath for 10 min, and then centrifuged at 15,000 rpm for 30 min at 4°C. Finally, the supernatant was collected and carefully transferred to a fresh RNase-free tube and stored at -80°C for later use.

[0061] Step 2, extension of telomerase primer sequence: take 20uL 50uM telome...

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Abstract

The invention discloses a telomerase activity detection kit and a detection method, and belongs to the technical field of nanopore sensing. According to the method, telomeres are extended and amplified with a specific sequence (TTAGGG) n by using telomerase under the action of a primer to obtain a DNA sequence rich in G basic groups, the DNA sequence is combined with potassium ions and hemin to form a G-quadruplex structure, and the generated G-quadruplex has peroxidase-like activity, can catalyze hydrogen peroxide reaction and perform morphology regulation on precious metal nanoparticles, and has a good application prospect. The nanoparticle product before and after the reaction is subjected to signal detection in the solid nanopore, so that the high-sensitivity detection on the telomerase activity is realized. According to the invention, the active reaction of telomerase and nanoparticle morphology regulation are combined, labeling is not needed, the reaction is efficient, extension of telomeres is converted into morphology change of single nanoparticles, the method has the effects of signal amplification and flux improvement, and high-sensitivity detection of telomerase activity under low-sample and low-concentration conditions is facilitated.

Description

technical field [0001] The invention provides a telomerase activity detection kit and detection method, combined with telomerase extension specific sequence to form a catalase-like reaction and gold nanorod etching technology, the telomerase activity can be detected through the engraving of gold nanorods. The shape changes during the erosion process, and signal detection is performed in the nanopore, thereby realizing the highly sensitive detection of telomerase activity by the solid-state nanopore, which belongs to the field of nanopore sensor detection. Background technique [0002] Telomere is a special structure at the end of eukaryotic chromosomes. During the DNA replication process of each cell division cycle, telomere will gradually shorten. When the telomere shortens to a critical length, it will lead to programmed cell death. Telomerase is a reverse transcriptase that can maintain the telomere length of chromosomes. It achieves the purpose of maintaining telomere le...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/686C12Q1/48G01N27/327G01N27/49
CPCC12Q1/686C12Q1/485G01N27/3277G01N27/3278G01N27/49G01N2333/9128C12Q2521/107C12Q2533/101C12Q2563/137C12Q2563/155C12Q2565/607C12Q2565/631
Inventor 武灵芝曾祥杰王宇朋王润雨翁丽星
Owner NANJING UNIV OF POSTS & TELECOMM
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