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EGFR gene 20 exon T790M and C797S mutation detection primers, probes and method

A technology of T790M and C797S, applied in the fields of biology and nucleic acid detection, can solve the problem of low frequency of tumor mutation, achieve good quenching effect, low fluorescence background, and reduce interference.

Inactive Publication Date: 2017-08-22
上海捷易生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The proportion of ctDNA in cfDNA fluctuates widely (0.01% to 90%), affected by tumor stage, metastasis, etc., most of which are on the order of a few thousandths, resulting in a very low frequency of tumor mutations that can be detected in cfDNA

Method used

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  • EGFR gene 20 exon T790M and C797S mutation detection primers, probes and method
  • EGFR gene 20 exon T790M and C797S mutation detection primers, probes and method
  • EGFR gene 20 exon T790M and C797S mutation detection primers, probes and method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Mutation detection of EGFR gene exon 20 T790M and C797S

[0032] 1. Design and synthesize primers and probes

[0033] The DNA sequence of the human EGFR (NM_005228) gene was searched from NCBI, and primers and corresponding Taqman probes for amplifying the T790M and C797S sites of exon 20 of the EGFR gene were designed and synthesized.

[0034] Wherein, the primers include a forward primer and a reverse primer, and the sequences are respectively:

[0035] Forward primer: 5'-GCCTGCTGGGCATCTG-3' (SEQ ID NO: 1)

[0036]Reverse primer: 5'-TCTTTGTGTTCCCGGACATAGTC-3' (SEQ ID NO:2)

[0037] The probes include a wild-type probe combined with a wild-type template DNA and a mutant probe combined with a mutant template DNA, and the sequences are respectively (wherein, VIC and FAM are fluorescent reporter groups, and MGB is a minor groove binder , NFQ is a non-fluorescent quencher group):

[0038] Wild-type probe for detecting T790M site: 5'-VIC-ATGAGCTGCGTGATGAG-MGB-...

Embodiment 2

[0073] Embodiment 2 detection sensitivity (mutation detection rate) experiment

[0074] 1) The mutant plasmid constructed in Example 1 and the wild-type plasmid were mixed according to the following ratios: 1:20; 1:50; 1:100; 1:200; 1:500; 1:1000; 1:5000.

[0075] 2) Prepare PCR amplification reaction solution according to the following ratio: 10 μl of 2X ddPCR master mix, 1.8 μl of 10 μM forward primer, 1.8 μl of 10 μM reverse primer, 0.5 μl of 10 μM wild-type probe, 0.5 μl of 10 μM mutant probe, DNA Template 5μl (30ng, 10000 copies), add water to 20μl.

[0076] 3) Add the prepared PCR amplification reaction liquid into the 8-channel micro-droplet making plate, then add 70 μl of micro-droplet generating oil, and prepare the PCR micro-droplet reaction liquid with QX200 micro-droplet generator. Add the prepared PCR droplet reaction solution into a 96-well PCR plate, and heat seal the film with a sealing film plate.

[0077] 4) Put the 96-well PCR plate into the PCR instrument...

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Abstract

The invention discloses a primer pair for specific amplification of EGFR gene 20 exon T790M and C797S loci. The sequence of the primer pair is shown in SEQ ID No:1-2. The invention further discloses a probe set for specific detection of the T790M and C797S loci. The probe set comprises a wild type probe and a mutant type probe, and the sequences are shown in SEQ ID No:3-7. The invention further discloses a reagent kit comprising the primer pair and the probe set, and a method for adopting the primer pair and the detection set for detecting EGFR gene 20 exon T790M and C797S mutation. By designing the specific primers and probes, the 3' end of the probes is connected with a minor groove binder and a non-fluorescent quenching group, the interference of background signals is lowered, hybridization of the probes and a template is stabilized, a Tm value of the probes is increased, and therefore the mutation detection sensitivity is improved.

Description

technical field [0001] The present invention relates to the field of biology, in particular to the field of nucleic acid detection, more specifically, to the detection of T790M and C797S mutations in exon 20 of EGFR gene. Background technique [0002] At present, lung cancer is recognized as the "king of cancer". In my country, the mortality rate of lung cancer is the fastest rising rate among cancers. About 80% of lung cancer is non-small cell lung cancer (Non-Small Cell Lung Cancer, NSCLC). Although traditional chemotherapy and radiotherapy are the mainstream in the treatment of lung cancer, due to the lack of specificity, while achieving curative effect, they also bring relatively large side effects. NSCLC is more sensitive to it. Therefore, people urgently need to study new treatment methods. Among them, molecular targeted therapy has received more and more attention, and drugs that target key enzymes in signal transduction pathways related to tumor cell proliferation ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6851C12Q1/6858C12Q2561/101C12Q2563/159C12Q2545/113
Inventor 陈艳李艳艳欧恩智陈珺朱智
Owner 上海捷易生物科技有限公司
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