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RET fusion gene ARMS (amplification refractory mutation system) fluorescent quantitative PCR typing detection kit

A fusion gene and fluorescence quantitative technology, which is applied in the determination/inspection of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., can solve problems such as the inability to obtain amplification products, and achieve simplification of experiments, improvement of signal-to-noise ratio, and optimization steps. simple effect

Active Publication Date: 2016-04-20
ANHUI DAJIAN MEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

That is, during PCR amplification, the extension of the primer starts from its 3' end, and the extension requires that the bases at the 3' end of the primer must be completely paired with the template, only in this way can the primer be extended and the amplification can proceed If the 3' end of the primer cannot be paired with the template, the extension of the primer will be blocked and the corresponding amplification product will not be obtained.

Method used

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  • RET fusion gene ARMS (amplification refractory mutation system) fluorescent quantitative PCR typing detection kit
  • RET fusion gene ARMS (amplification refractory mutation system) fluorescent quantitative PCR typing detection kit
  • RET fusion gene ARMS (amplification refractory mutation system) fluorescent quantitative PCR typing detection kit

Examples

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Effect test

Embodiment 1

[0120] Example 1: Detection of RET fusion gene KR1 (KIF5B exon 15 / RET exon 12 fusion)

[0121] Use the fluorescent quantitative reaction solution A in the invented RET fusion gene ARMS fluorescent quantitative PCR typing detection kit for detection; then optimize the reaction system for ARMS-qPCR detection: the reaction system is 25μL, fluorescent quantitative reaction solution A (primers, probes) Mixture, concentration of 5uM) 1μL, sample template DNA 2μL (100-300ng / μL), 2*TaqmanuniversalPCRMasterMix (purchased from American Applied Biology) 15μL, ddH 2 O7μL.

[0122] PCR reaction conditions: 95°C pre-denaturation for 30 seconds; 95°C for 15 seconds, 62°C for 20 seconds, 20 cycles of amplification reaction; and 95°C for 15 seconds, 60°C for 34 seconds, and 40 cycles of amplification reaction.

[0123] At the same time, in the fluorescence quantitative test, while testing the sample, it is also necessary to set the sample reference product to determine the validity of the test, such ...

Embodiment 2

[0124] Example 2: Detection of RET fusion gene KR2 (KIF5B exon 16 / RET exon 12 fusion)

[0125] Use the fluorescent quantitative reaction solution A in the invented RET fusion gene ARMS fluorescent quantitative PCR typing detection kit for detection; then optimize the reaction system for ARMS-qPCR detection: the reaction system is 25μL, fluorescent quantitative reaction solution A (primers, probes) Mixture, concentration of 5uM) 1μL, sample template DNA 2μL (100-300ng / μL), 2*TaqmanuniversalPCRMasterMix (purchased from American Applied Biology) 15μL, ddH 2 O7μL.

[0126] PCR reaction conditions: 95°C pre-denaturation for 30 seconds; 95°C for 15 seconds, 62°C for 20 seconds, 20 cycles of amplification reaction; and 95°C for 15 seconds, 60°C for 34 seconds, and 40 cycles of amplification reaction.

[0127] At the same time, in the fluorescence quantitative test, while testing the sample, it is also necessary to set the sample reference product to determine the validity of the test, such ...

Embodiment 3

[0128] Example 3: Detection of RET fusion gene KR3 (KIF5B exon 23 / RET exon 12 fusion)

[0129] Use the fluorescent quantitative reaction solution A in the invented RET fusion gene ARMS fluorescent quantitative PCR typing detection kit for detection; then optimize the reaction system for ARMS-qPCR detection: the reaction system is 25μL, fluorescent quantitative reaction solution A (primers, probes) Mixture, concentration of 5uM) 1μL, sample template DNA 2μL (100-300ng / μL), 2*TaqmanuniversalPCRMasterMix (purchased from American Applied Biology) 15μL, ddH 2 O7μL.

[0130] PCR reaction conditions: 95°C pre-denaturation for 30 seconds; 95°C for 15 seconds, 62°C for 20 seconds, 20 cycles of amplification reaction; and 95°C for 15 seconds, 60°C for 34 seconds, and 40 cycles of amplification reaction.

[0131] At the same time, in the fluorescence quantitative test, while testing the sample, it is also necessary to set the sample reference product to determine the validity of the test, such ...

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Abstract

The invention discloses an RET fusion gene ARMS (amplification refractory mutation system) fluorescent quantitative PCR typing detection kit and relates to a primer, a probe and a detection kit for detecting 7 common KIF5B-RET fusion gene variants and 1 RET fusion gene type ARMS-qPCR, wherein the RET fusion gene mutation comprises eight fusion variants including KR1(K15:R12), KR2(K16:R12), KR3(K23:R12), KR4(K24:R8), KR5(K22:R12), KR6(K24:R11), KR7(K15:R11) and newly discovered NR8(N6:R12). In the invention, the detection is fast, efficient and sensitive; the result interpretation is very clear and visual, and the result is reliable and peculiar; and by adopting the primer, probe and kit provided by the invention, 8 RET fusion gene mutations can be effectively detected.

Description

Technical field [0001] The invention belongs to the field of molecular biology gene detection, and relates to a RET fusion gene ARMS fluorescent quantitative PCR typing detection kit, and specifically relates to 7 most common KIF5B-RET fusion gene variants and 1 newly discovered RET fusion genotype ARMS-qPCR The detection kit is suitable for the detection of RET fusion gene mutations in lung adenocarcinoma. Background technique [0002] Lung cancer is the tumor with the highest mortality rate in the world, and its mortality rate is also the first among tumors, mainly including small cell lung cancer (SCLC) and non-small cell lung cancer (non-small cell lung cancer, NSCLC). NSCLC includes squamous cell carcinoma. , Adenocarcinoma, adenosquamous carcinoma, large cell carcinoma, carcinoid, etc. In my country, the mortality rate of lung cancer has ranked first among tumor mortality rates. Among them, the incidence rate of NSCLC accounts for 80% of all lung cancer incidence rates, an...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6827C12Q1/6886C12Q2600/156C12Q2563/107C12Q2545/114C12Q2531/113
Inventor 钟明李香梅
Owner ANHUI DAJIAN MEDICAL TECH CO LTD
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