Telomerase activity detection kit and detection method

A technology of activity detection and telomerase, applied in measurement devices, instruments, scientific instruments, etc., can solve problems such as the inability to detect telomerase activity

Active Publication Date: 2018-12-07
THE SECOND PEOPLES HOSPITAL OF SHENZHEN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this patent method still cannot realize the detection of telomerase activity

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

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preparation example Construction

[0051] II. Preparation and characterization of quantum dot ratiometric fluorescent probes

[0052] 1. Preparation method

[0053] According to previous literature (Mao G., Q.Cai, F.Wang, C.Luo, X.Ji&Z.He, One-Step Synthesis of Rox-DNA Functionalized CdZnTeS Quantum Dots for the Visual Detection of Hydrogen Peroxide and Blood Glucose. Anal Chem 89, 11628-11635(2017)), Preparation of CdZnTeS quantum dot ratiometric fluorescent probes by a one-pot hydrothermal method. The specific preparation steps are as follows.

[0054] A. Design and synthesis of DNA sequence modified by Rox dye and phosphorothioate

[0055] The DNA sequence for the design of Rox dyes co-modified with phosphorothioate is as follows:

[0056] G*G*G*G*G*G*G*G*AAAAAAAACCTTCCTCCGCAATACTCCCCAGGTAAA-Rox (5'→3', where * indicates phosphorothioate modification). That is, the modified DNA sequence is modified with phosphorothioate at the 5' end and Rox dye at the 3' end, or it can be modified with phosphorothioate ...

Embodiment 1

[0087] In this example, the telomerase sample obtained from HeLa cells is used to illustrate the quantitative detection method of telomerase activity of the present invention, wherein the color reaction is carried out in Tris buffer solution of Rox-CdZnTeS quantum dot ratio fluorescent probe.

[0088] (1) Obtain telomerase samples

[0089] HeLa cells were cultured in DMEM medium containing 10% heat-inactivated FBS. After harvesting cells with trypsin, 1 x 10 6 The cells were collected into 1.5mL EP tubes and centrifuged at 1000rpm for 10 minutes. Cells were washed once with phosphate-buffered saline (pH=7.4), centrifuged again, and dispensed in 200 μL of ice-cold 1×CHAPS lysis buffer. Then, the cells were incubated on ice for 30 minutes, centrifuged at 12000 rpm for 20 minutes at 4°C, and the obtained supernatant was the telomerase sample. Transfer the supernatant to a fresh tube and store at -80°C.

[0090] (2) Formation of hemin / G-quadruplex

[0091] First, add 1 μL of ...

Embodiment 2

[0099] Embodiment 2: test paper sensor method

[0100] The steps (1) to (3) of this embodiment are the same as those of Embodiment 1, but in step (4), the color reaction is carried out with a test paper sensor. The differences from Embodiment 1 are mainly described below.

[0101] Specifically, before step (4), the Rox-CdZnTeS quantum dot ratio fluorescent probe was dissolved in Tris buffer (10uL) in advance to form a Rox-CdZnTeS quantum dot ratio fluorescent probe solution (200nM), and the test paper Immerse in the Rox-CdZnTeS quantum dot ratio fluorescent probe solution, and air-dry at room temperature for 5 minutes to form a test paper sensor.

[0102] Step (4): Take 10 uL of the reacted mixed system in step (4) with a microburette, drop it on the test paper sensor, react with the Rox-CdZnTeS quantum dot ratio fluorescent probe on the test paper sensor for 5 minutes, and display the fluorescent color.

[0103] Step (5): Production of standard color cards

[0104] Since th...

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Abstract

The invention provides a telomerase activity detection kit, which includes: a telomerase substrate primer, dNTPs, a telomerase reverse transcription buffer solution, hemin, hydrogen peroxide, and a quantum dot ratio fluorescence probe. Specifically, the quantum dot ratio fluorescence probe includes a quantum dot part and a dye part, the quantum dot part and the dye part emit different colors of fluorescence, and hydrogen peroxide can quench the fluorescence emitted by the quantum dot part, but cannot quench the fluorescence emitted by the dye part. The quantum dot part and the dye part are compounded through thiophosphate modified DNA. The quantum dot is CdZnTeS quantum dot, and the dye is part is Rox. The invention also provides a telomerase activity detection quantitative detection method for nondiagnostic therapy purpose. The detection kit and detection method provided by the invention can realize quantitative detection and visual discrimination of telomere.

Description

technical field [0001] The invention relates to the field of biological detection, in particular to a method for quantitative detection of telomerase activity for the purpose of non-diagnosis and treatment. Background technique [0002] Human telomerase is a nucleoprotein reverse transcriptase that consists of three components: telomerase reverse transcriptase, telomerase RNA component, and associated proteins. Telomerase, responsible for adding the telomere repeat sequence (TTAGGG)n to the ends of human chromosomes, is overexpressed in 85% of malignant tumor cells but not in normal tissues. Therefore, telomerase can be used as a specific tumor marker, and effective detection of telomerase activity has great value in cancer diagnosis and treatment. [0003] Telomere repeat amplification (TRAP) has been considered as the gold standard method for detecting telomerase. Despite the high sensitivity of this most commonly used method, it still has many disadvantages such as time...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/573
CPCG01N33/573
Inventor 马英新黄卫人
Owner THE SECOND PEOPLES HOSPITAL OF SHENZHEN
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