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Telomerase activity detection kit and telomerase activity detection method

A technology for activity detection and telomerase, applied in the field of biochemical detection, can solve the problems of complex operation, false positive, time-consuming, etc.

Pending Publication Date: 2020-06-12
THE SECOND PEOPLES HOSPITAL OF SHENZHEN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although TRAP and its improved detection methods have high sensitivity and good specificity, these polymerase chain reaction (PCR)-based methods also have obvious disadvantages, such as complicated operation, time-consuming, false positives, and inability to detect in situ

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Embodiment 1: Telomerase activity detection kit

[0093] Add 20 μL volume of telomerase substrate primer 5′-AAT CCG TCG AGC AGA GTT-3’, 5 μL volume of dNTPs and 875 μL volume of telomerase reverse transcription buffer (20 mM Tris-HCl (pH=8.3), 1.5 mM MgCl 2 , 63mMKCl, 0.005% Tween 20, BSA 0.1mg / mL) into the first reagent container with a volume of 2mL. 1 mg of the QD-BHQ2 quantum dot molecular beacon prepared as described in the experimental part above was dissolved in 300 μL of Tris buffer (pH=8.0) to prepare a QD-BHQ2 quantum dot molecular beacon solution. A 100 μL volume of this QD-BHQ2 quantum dot molecular beacon solution was loaded into a second 0.5 mL volume reagent container. Fill 100 µL of telomerase cell extract standard solution into a third 0.5 mL volume reagent container.

[0094] Putting the first reagent container, the second reagent container and the third reagent container together with the instructions for detecting telomerase activity into a packag...

Embodiment 2

[0095] Example 2: In vitro detection of telomerase activity

[0096] Fresh urine samples (200 mL) were collected and centrifuged at 1000 rpm for 10 minutes at 4°C to collect the precipitate. The pellet was washed with PBS (pH 7.4) and centrifuged at 1800 rpm for 5 minutes at 4°C. The pellet was then resuspended in 2 mL of ice-cold 1× CHAPS lysis buffer. The mixture was incubated on ice for 30 minutes and then centrifuged at 12000 rpm for 20 minutes at 4°C. Transfer the supernatant containing telomerase to a new 1.5 mL EP tube and store at -80 °C until use.

[0097] Take 1 μL volume of the supernatant, and take 0.2 μL volume of telomerase substrate primer, 0.05 μL volume of dNTPs, and 8.75 μL volume of telomerase reverse transcription buffer in the telomerase activity detection kit of Example 1, and carry out Extension reaction (temperature 37°C, time 1h). Then, take 1 μL of the reaction system, add 1 μL of the QD-BHQ2 quantum dot molecular beacon solution in the telomerase...

Embodiment 3

[0099] Example 3: Extracellular detection of telomerase activity

[0100] will be 5×10 5HeLa cells were plated in 20mm confocal dishes for 24 hours. Then take 10 μL volume of the telomerase substrate primer in the telomerase activity detection kit of Example 1. 10 μL Lipofectamine 3000 in 250 μL Opti-MEM was added to transfect the cells for 1 hour at 37° C., after which the Opti-MEM transfection mixture was removed. Take 20 μL of the quantum dot molecular beacon in the telomerase activity detection kit of Example 1, add it to 2 mL of fresh medium, then add the mixture to the cells, and incubate in the confocal culture dish at 37° C. for 4 hours. The supernatant was then replaced with fresh Opti-MEM in a volume of 2 mL under an UltraView Vox spinning disk confocal laser scanning system (PerkinElmer) with a Nikon Ti-e microscope (60x objective) and a cell culture environment chamber (Tokai Hit) Observe the cells. Observe whether there is red fluorescence excited at 580nm (ex...

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Abstract

The invention relates to a telomerase activity detection kit, which comprises a telomerase substrate primer, dNTPs, a telomerase reverse transcription buffer solution and a quantum dot molecular beacon, wherein the quantum dot molecular beacon is composed of an oligonucleotide stem-loop, a quantum dot fluorophore and a quenching group, the quantum dot fluorophore is connected with the 5' end of the sequence of the oligonucleotide stem-loop, the quenching group is connected with the 3' end of the sequence of the oligonucleotide stem-loop, the quantum dot fluorophore is preferably CdTe:Zn<2+> quantum dots, the quenching group is preferably BHQ2, and the quantum dot molecular beacon is preferably QD-BHQ2. Accordinto the invention, the quantum dot molecular beacon does not need excessive chemical modification, does not need signal amplification, has excellent biocompatibility, fluorescence recovery, detection sensitivity and light stability, and does not have cytotoxicity; and the telomerase activity detection kit can be directly used for sensitive visual detection of telomerase activity.

Description

technical field [0001] The invention relates to the technical field of biochemical detection, in particular to a telomerase activity detection kit and a telomerase activity detection method, and the method is particularly applicable to the detection of telomerase activity for non-diagnostic and therapeutic purposes. Background technique [0002] Telomerase is a reverse transcriptase that plays an important role in maintaining telomere length by adding repeating units (TTAGGG)n to the ends of chromosomes. It is well known that during cell division in most normal cells, telomeres gradually shorten without detectable telomerase activity. Telomerase expression is related to tumorigenesis, so the detection of telomerase activity is also an important criterion for early diagnosis of cancer. Several methods have been developed to detect telomerase activity, among which the Telomere Repeat Amplification Protocol (TRAP) is the most commonly used and has become a gold method. Althou...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/48G01N21/64
CPCC12Q1/48G01N21/6428G01N2021/6439
Inventor 马英新黄卫人钟玉城
Owner THE SECOND PEOPLES HOSPITAL OF SHENZHEN
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