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Optimization method of BRAF gene V600E mutation digital PCR detection system and detection product

An optimization method and digital technology, applied in the fields of biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of insensitive and accurate detection of plasma cell-free DNA mutation information, low cfDNA content, etc., and achieve low cost and results. accurate 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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  • Application Information

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Problems solved by technology

However, the content of ctDNA in cfDNA is extremely low, less than 1%, and traditional detection methods are not sensitive and accurate in detecting the mutation information contained in plasma cell-free DNA

Method used

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  • Optimization method of BRAF gene V600E mutation digital PCR detection system and detection product
  • Optimization method of BRAF gene V600E mutation digital PCR detection system and detection product
  • Optimization method of BRAF gene V600E mutation digital PCR detection system and detection product

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Embodiment 1

[0034] The BRAF gene V600E mutation digital PCR detection kit of this embodiment includes upstream primers (BRAF-V600E-F), downstream primers (BRAF-V600E-R), wild-type probes (BRAF-V600E-wt) and mutant probes (BRAF-V600E-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.

BRAF-V600E-F

CTACTGTTTTCCTTTACTTACTACACCTCAGA

1

BRAF-V600E-R

ATCCAGACAACTGTTCAAACTGATG

2

BRAF-V600E-wt

CTAGCTACAGTGAAATC

3

BRAF-V600E-mt

TAGCTACAGAGAAATC

4

[0038] The 5' end of the ...

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Abstract

The invention relates to an optimization method of a BRAF gene V600E 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 BRAF gene V600E 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 BRAF gene V600E 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 BRAF gene V600E mutation and an optimization method thereof, belonging to the field of biotechnology. Background technique [0002] The BRAF gene is a member of the RAF gene family and is a downstream gene of the RAS pathway. There are two other genes in this gene family: ARAF and CRAF. The BRAF gene is located on chromosome 7q34 with a length of about 190kb and consists of 18 exons. The BRAF gene plays an important role in the Ras-Raf-MEK-ERK signal transduction pathway, and its encoded protein has three conserved regions, CR1, CR2 and CR3, among which CR1 and CR2 have the function of regulating catalytic activity, and CR3 is a kinase region, which is ATP activation domain and binding site. [0003] The BRAF gene acts through the serine / threonine protein kinase in the silk protein kinase pathway, and connects the receptor and RAS protein on the cell surface to the transcrip...

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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