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Optimization method of EGFR gene T790M mutation digital PCR detection system and detection product

An optimization method and digital technology, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problem that the sensitivity of ordinary ARMS-PCR cannot meet the detection requirements, and achieve the effect of accurate results

Pending Publication Date: 2019-02-01
上海赛安生物医药科技股份有限公司
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Problems solved by technology

However, because the ctDNA content in plasma is less than 1% and highly fragmented (180bp), the sensitivity of ordinary ARMS-PCR can reach up to 1% and cannot meet the detection requirements

Method used

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  • Optimization method of EGFR gene T790M mutation digital PCR detection system and detection product
  • Optimization method of EGFR gene T790M mutation digital PCR detection system and detection product
  • Optimization method of EGFR gene T790M mutation digital PCR detection system and detection product

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Embodiment 1

[0034] The EGFR gene T790M mutation digital PCR detection kit of this embodiment includes upstream primer (EGFR-T790M-F), downstream primer (EGFR-T790M-R), wild-type probe (EGFR-T790M-wt) and mutant probe (EGFR-T790M-mt), digested normal human gDNA and digested mutant plasmid.

[0035] The primers and probes are self-designed and optimized through multiple combinations, and the primers and probes include the homologous region of the mutant fragment inserted in the mutant plasmid. The primers and probes were synthesized by Shanghai Bailige Biotechnology Co., Ltd. The nucleotide sequences of the primers and probes are shown in Table 1.

[0036] Table 1 Primer Probe Sequence List

[0037] name

Sequence(5'—3')

Seq No.

EGFR-T790M-F

CCTCACCCTCCACCGTGCA

1

EGFR-T790M-R

GTCTTTGTGTTCCCGGACATAGT

2

EGFR-T790M-wt

ATGAGCTGCGTGATGAG

3

EGFR-T790M-mt

ATGAGCTGCATGATGAG

4

[0038] The 5' end of the wild-type probe (E...

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Abstract

The invention relates to an optimization method of a EGFR gene T790M mutation digital PCR detection system, wherein the detection system includes upstream primers, downstream primers, wild-type probes, mutant-type probes, a wild-type template and a mutant-type template; the wild-type template is normal human gDNA after enzyme digestion, and the mutant-type template is a mutant plasmid inserted with a EGFR gene T790M mutant fragment after enzyme digestion; a standard substance is prepared from the mutant-type template and the wild-type template according to a certain proportion of copy number;reaction is performed with a medium mutation frequency standard substance as a template by digital PCR, a data statistical graph is prepared according to the reaction data, and a wild-type fluorescentregion and a mutant-type fluorescent region are selected. Reaction is performed with the wild-type template as the template by digital PCR, and the background threshold value is determined. A detection product optimized by the optimization method of the EGFR gene T790M mutation digital PCR detection system has higher accuracy degree.

Description

technical field [0001] The invention relates to a digital PCR detection product for detecting human EGFR gene T790M mutation and an optimization method thereof, belonging to the field of biotechnology. Background technique [0002] Lung cancer is the malignant tumor with the highest morbidity and mortality worldwide. Among lung cancer patients, the incidence of non-small cell lung cancer (NSCLC) is 85%, and more than 70% of them are in the middle and advanced stages. Among them, the epidermal growth factor receptor gene (EGFR) is the main driver gene of lung adenocarcinoma in my country. [0003] Epidermal growth factor receptor (EGFR) belongs to the tyrosine kinase receptor, which runs through the cell membrane, and its intracellular area is divided into its kinase active area. The activated EGFR monomer is transformed into an activated dimer, which can guide downstream phosphorylation and induce cell proliferation. The tyrosine kinase active region of the EGFR gene cont...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/6858C12Q1/6851
CPCC12Q1/6886C12Q1/6851C12Q1/6858C12Q2600/156C12Q2531/113C12Q2563/107C12Q2545/114
Inventor 赵新泰王明潘文健
Owner 上海赛安生物医药科技股份有限公司
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