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Optimization method of PIK3CA gene H1047R 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 low cfDNA content, insensitive and accurate detection of plasma cell-free DNA mutation information, etc., and achieve 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, 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 PIK3CA gene H1047R mutation digital PCR detection system and detection product
  • Optimization method of PIK3CA gene H1047R mutation digital PCR detection system and detection product
  • Optimization method of PIK3CA gene H1047R mutation digital PCR detection system and detection product

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Embodiment 1

[0034] The PIK3CA gene H1047R mutation digital PCR detection kit of this embodiment includes upstream primer (PIK3CA-H1047R-F), downstream primer (PIK3CA-H1047R-R), wild-type probe (PIK3CA-H1047R-wt) and mutant probe (PIK3CA-H1047R-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.

PIK3CA-H1047R-F

GCAAGAGGCTTTGGAGTATTTCATG

1

PIK3CA-H1047R-R

GCTGTTTAATTGTGTGGAAGATCCAA

2

PIK3CA-H1047R-wt

CCACCATGATGTGCATC

3

PIK3CA-H1047R-mt

CACCATGACGTGCATC

4

[0038] T...

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Abstract

The invention relates to an optimization method of a PIK3CA gene H1047R 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 insertedwith a PIK3CA gene H1047R 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 fluorescent region 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 PIK3CA gene H1047R mutation digital PCR detection system has higher accuracy degree.

Description

technical field [0001] The invention relates to a digital PCR detection product and an optimization method for detecting human PIK3CA gene H1047R mutation, belonging to the field of biotechnology. Background technique [0002] Phosphatidylinositol-3-kinase catalytic subunit α (PIK3CA) gene was first identified as a 34kb gene on chromosome 3q26.3 by in situ hybridization. PIK3CA consists of 20 exons and encodes the p110α catalytic subunit of class I phosphatidylinositol-3-kinase (PI3K). PI3K is a class of phosphorylated inositol lipid 3-hydroxykinases, known as the PI3Ks family. Class I PI3Ks are heterodimers composed of a p110 catalytic subunit and a p85 regulatory subunit, which are activated by growth factor receptors and insulin receptors. PI3Ks are usually expressed in the cytoplasm and produce proteins with second messenger functions. Inositol substances play an important role in cell morphology, vitality, invasiveness, apoptosis and cell cycle regulation. The PI3K-A...

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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