Probe method detecting human TERT gene promoter mutation and reagent kit thereof

A promoter and human detection technology, applied in the direction of determination/inspection of microorganisms, biochemical equipment and methods, etc.

Pending Publication Date: 2016-02-17
GENETRON HEALTH (BEIJING) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing TERT gene detection can only analyze the expression of TERT gene by in situ hybridization, which must be based on cell morphology and does not include the amplificat

Method used

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  • Probe method detecting human TERT gene promoter mutation and reagent kit thereof
  • Probe method detecting human TERT gene promoter mutation and reagent kit thereof
  • Probe method detecting human TERT gene promoter mutation and reagent kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Composition of the kit

[0047] The kit of the present invention is a kit for detecting mutations of TERTC228T and TERTC250T gene promoters, including: TERTC228TPCR reaction mixture, TERTC250TPCR reaction mixture, weak positive control solution and blank control solution, wherein:

[0048] (1) TERTC228TPCR reaction mixture is to add various substances at the following concentrations into the reaction premix TakaraPremixExTaq (ProbeqPCR):

[0049] TERT C228T upstream specific primer

3μM

TERT Universal Primer

3μM

TERT Universal Probe

3μM

internal reference upstream primer

2μM

Downstream primers for internal reference

2μM

internal reference probe

2μM

dUTP

0.2μM

uracil DNA glycosylase

0.01U / μl

[0050] (2) TERTC250TPCR reaction mixture is to add various substances at the following concentrations into the reaction premix TakaraPremixExTaq (ProbeqPCR):

[0051] TERT C2...

Embodiment 2

[0088] Embodiment 2 Fluorescent PCR method contrasts the methodology verification of Sanger

[0089] Fluorescent PCR detection was carried out according to the method in Example 1, and Sanger verification was carried out at the same time. The verification data and results were as follows: 95 cases of glioma patients were randomly selected from frozen tissue samples to compare qPCR with the Sanger method in parallel, and it was obtained that qPCR and the Sanger method were consistent Rate verification results. According to the research results of clinical samples of glioma patients obtained so far, a total of 93 cases of effective detection data have been obtained. Compared with the Sanger detection methodology, the verification results show that the consistency rate between qPCR detection and Sanger verification is 100% for C228T and 100% for C250T.

[0090]The samples used for comparison were derived from glioma samples, including: anaplastic oligodendroglioma, astrocytoma,...

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Abstract

The invention discloses a probe method detecting human TERT gene promoter mutation and a reagent kit thereof. The method includes the following steps that firstly, human genome DNA is added in a TERT C228T PCR reaction mixed solution so that a C228T reaction system can be formed and fluorescence quantitative PCR amplification can be performed, and/or human genome DNA is added in a TERT C250T PCR reaction mixed solution so that a C250T reaction system can be formed and fluorescence quantitative PCR amplification can be performed; secondly, a result is judged, wherein analysis is performed according to the mutation Ct value of PCR amplification. The reagent kit comprises the TERT C228T PCR reaction mixed solution and/or the TERT C250T PCR reaction mixed solution. The probe method and the reagent kit provide a high-speed, high-sensitivity and high-specificity TERT gene promoter mutation detection system clinically.

Description

technical field [0001] The invention relates to a method for detecting gene mutation and its kit, in particular to a real-time fluorescent quantitative PCR Taqman probe method for detecting human TERT gene promoter mutation and its kit. Background technique [0002] Telomerase is a ribonucleoprotein complex composed of RNA and protein, belonging to reverse transcriptase, which is closely related to the regulation mechanism of telomeres and plays a key role in maintaining cell immortalization and carcinogenesis. Telomerase reverse transcriptase (telomerase reverse transcriptase, TERT) is an important catalytic part of telomerase. The TERT gene is located on human chromosome 5, and the two promoter mutation sites of the gene are 1,295,228C>T and 1,295,250C>T (referred to as C228T and C250T, respectively) are particularly common, and these two mutations have been shown to enhance transcriptional activity of the TERT promoter. Mutations in the TERT promoter region do not ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6827C12Q1/686C12Q1/6886C12Q2561/113C12Q2563/107C12Q2545/114C12Q2561/101C12Q2531/113
Inventor 阎海焦雨辰王晓月王思振熊梓锴
Owner GENETRON HEALTH (BEIJING) CO LTD
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