Optimization method of EGFR gene L858R mutation digital PCR detection system and detection product

An optimization method and digital technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of short cfDNA fragments, detection sensitivity can only reach 1%, low ctDNA content, etc., and achieve accurate results. Effect

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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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still some challenges in detecting EGFR gene mutations using cfDNA
The content of cfDNA varies from person to person, and most people have a low content; cfDNA fragments are short, with a max

Method used

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  • Optimization method of EGFR gene L858R mutation digital PCR detection system and detection product
  • Optimization method of EGFR gene L858R mutation digital PCR detection system and detection product
  • Optimization method of EGFR gene L858R mutation digital PCR detection system and detection product

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Embodiment 1

[0034] The EGFR gene L858R mutation digital PCR detection kit of this embodiment includes upstream primer (EGFR-L858R-F), downstream primer (EGFR-L858R-R), wild-type probe (EGFR-L858R-wt) and mutant probe (EGFR-L858R-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-L858R-F

GCAGCATGTCAAGATCACAGAT

1

EGFR-L858R-R

CCTCCTTCTGCATGGTATTCTTTCT

2

EGFR-L858R-wt

AGTTTGGCCAGCCCAA

3

EGFR-L858R-mt

AGTTTGGCCCGCCCAA

4

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

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Abstract

The invention relates to an optimization method of a EGFR gene L858R 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 L858R 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 L858R 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 L858R mutation and an optimization method thereof, belonging to the field of biotechnology. Background technique [0002] Worldwide, lung cancer is the leading cause of tumor death, seriously endangering human health. About 1.5 million people die from lung cancer every year in the world. The pollution of air pollution, the impact of smoking, etc. all further aggravate the occurrence of lung cancer. The morbidity and mortality of lung cancer rank first among all malignant tumors. Therefore, the early diagnosis and treatment of lung cancer has always been the end point of research and the key to the prevention and treatment of lung cancer. [0003] Epidermal growth factor receptor (EGFR) belongs to the tyrosine kinase receptor, runs through the cell membrane, and is divided into its kinase activity area inside the cell, and is a proto-oncogene. Epidermal growth fac...

Claims

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Application Information

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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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