Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Dual temperature rapid cycling fluorescence quota PCR method for detecting telomerase activity and kit

A fluorescence quantification, telomerase technology, applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems of interfering detection results, unavoidable non-specific products, etc.

Inactive Publication Date: 2011-10-19
TIANJIN MEDICAL UNIV
View PDF3 Cites 9 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] However, since the template sequence for PCR amplification in the TRAP method is the substrate sequence and multiple telomere repeat sequences of unequal length, the PCR reaction inevitably introduces non-specific products, which interfere with the detection results.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Dual temperature rapid cycling fluorescence quota PCR method for detecting telomerase activity and kit
  • Dual temperature rapid cycling fluorescence quota PCR method for detecting telomerase activity and kit
  • Dual temperature rapid cycling fluorescence quota PCR method for detecting telomerase activity and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Effect of anchored hairpin primers on background fluorescent signal for real-time detection

[0045] In this example, the different effects of anchor hairpin primers and hairpin primers on the background fluorescence of real-time quantitative detection were compared.

[0046] In the experiment, the protein extract of immortalized cells that was heat-treated to inactivate telomerase (for the treatment method of immortalized cell samples, see the example below) was used as a negative sample, and the anchored hairpin primer and the hairpin primer were used as downstream primers for fluorescence analysis. Quantitative PCR detection. Under the condition that the cycle number was set to 40, its effect on the background fluorescence detected by real-time quantification was compared. The reaction reagents and conditions are as follows:

[0047] Reagent: negative sample, 0.1 μg PP, 0.5 μg QP, 0.05 μg AHP, 1×TRAP buffer, 1.5 mM MgCl 2 , 50 μM dNTP, 2U Taq enzyme, reaction volu...

Embodiment 2

[0051] Determination of Quantitative Working Curve and TPG Value of DS / AHP-TRAP Kit

[0052] The present invention is prepared following kit:

[0053] Washing solution: Hepes-KOH (PH 7.5) 10mmol / L, MgCl 2 1.5mmol / L, KCl 10mmol / L, DTT 1mmol / L.

[0054] Preparation method:

[0055] (1) Dissolve 23.83 mg of Hepes in 10 mL of secondary deionized water, adjust the pH to 7.5 with concentrated KOH solution, and prepare a 10 mmol / L Hepes-KOH (PH 7.5) solution.

[0056] (2) MgCl 2 ·6H 2 O 1.52mg, KCl 3.72mg, DTT 0.772mg were dissolved in 5mL 10mmol / L Hepes-KOH (PH 7.5) solution and stored at -20°C.

[0057] Hepes (Free acid): High Pure Grade, Shanghai Sangon Bioengineering Co., Ltd., AMRESCO packaging.

[0058] DTT: High Pure Grade, Shanghai Sangon Bioengineering Co., Ltd., subpackaged by AMRESCO.

[0059] Cell lysate: Tris-HCl (PH 7.5) 10mmol / L, EGTA 1mmol / L, MgCl 2 1mmol / L, β-mercaptoethanol 5mmol / L, glycerin 10%, CHAPS 0.5%, PMSF 0.1mmol / L.

[0060] Preparation method:

...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention relates to a dual temperature rapid cycling fluorescence quota PCR method for detecting telomerase activity and a kit. The method provided by the invention comprises steps of: combining primers, which are the anchored hairpin primers (AHP), with a duplex scorpion primers (DS) to form a DS / AHP-TRAP system; detecting the activity of telomerase in immortalized cell protein extracts under the condition of dual temperature rapid cycling PCR; taking a telomerase extension product R6 containing 6 telomere repeat sequences as a quantitative standard substance, and drafting a quantitative curve of the extension product of the detected telomerase; comparing the quantitative standard curve of the immortalized cell sample and that of R6 to obtain an anastomosis graph of the detected telomerase activity quantitative curve and the telomerase extension product quantitative curve; supposing that a TPG value is a telomerase extension product generated by an immortalized cell and determining the number of R6 represented by the TPG value. The kit provided by the invention can be used to accomplish a real-time detection of telomerase activity in large quantities on the fluorescence quota PCR instrument in a simpler and faster manner, and has a wide prospect in the early diagnosis of malignant tumor and the screening of anticancer drugs.

Description

technical field [0001] The invention relates to a detection method and kit for double-temperature fast cycle fluorescence quantitative PCR telomerase activity. This method uses Anchored hairpin structural primer (AHP) and compound scorpion primer (Duplex scorpion primer, DS) on a fluorescent quantitative PCR instrument, using dual-temperature fast-cycle PCR amplification conditions to carry out telomeric Detection of enzyme activity. It is a kit for detecting telomerase activity with DS / AHP-TRAP (anchor hairpin primer and composite scorpion primer-telomeric repeat sequence amplification assay). Background technique [0002] Telomerase (Telomerase) is a broad-spectrum tumor marker, which is highly expressed in nearly 90% of cancers, but generally not expressed in benign, precancerous lesions and surrounding tissues. The determination of telomerase activity has directness, high sensitivity and strong specificity, and can be used as an independent indicator for early diagnosi...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/25
Inventor 黄艳萍刘照胜汤华
Owner TIANJIN MEDICAL UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com