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Kit and method for detecting mutation of EGFR gene

A detection kit and kit technology, applied in the fields of biotechnology and tumor diagnosis, can solve the problems of high detection sensitivity and difficulty in meeting detection requirements

Active Publication Date: 2018-12-07
DAAN GENE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Since the concentration of cfDNA in the blood of patients with advanced lung cancer is extremely low, it requires high detection sensitivity. Commonly used gene mutation detection methods include: Sanger sequencing method, high-resolution melting point curve analysis method, real-time fluorescent quantitative PCR method, and high-efficiency denaturation method. Liquid chromatography techniques are difficult to meet its detection requirements

Method used

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  • Kit and method for detecting mutation of EGFR gene
  • Kit and method for detecting mutation of EGFR gene
  • Kit and method for detecting mutation of EGFR gene

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Effect test

preparation example Construction

[0136] Step 2, preparation of dPCR system

[0137] Preparations before preparation of the dPCR system: Take out the specific primers and probes, Mastermix, Solution, sterile water, etc. in the kit, melt at room temperature, vortex and oscillate to mix, and then centrifuge for 10 seconds to prepare the dPCR system; the composition of the dPCR system is shown in Table 5. :

[0138] Table 5 dPCR system

[0139] Specific Primers and Probes

1μL

Mastermix

7.5μL

Solution

0.75 μL

sterile water

Make up to 11 μL

[0140] The nucleotide sequence information of the primer probe is as follows:

[0141] Nucleotide sequence information of primers and probes for detection of C797S site mutation in exon 20:

[0142]

[0143] Nucleotide sequence information of primers and probes for detecting T790M site mutation in exon 20:

[0144]

[0145] Nucleotide sequence information of the primer probe for detecting the 19del deletion site of exo...

Embodiment 1

[0170] This embodiment provides the composition, packaging and quantity (48 reactions / box) of a test kit for detecting EGFR gene mutation in patients with non-small cell lung cancer, as shown in Table 7,

[0171] Table 7 Components, packaging and quantity of the kit

[0172]

Embodiment 2

[0173] Embodiment 2: Sensitivity detection and mutation detection rate experiment

[0174] The gDNA control samples are nucleic acid samples containing EGFR gene 19del wild type, T790M wild type nucleic acid, L858R wild type nucleic acid and C797S wild type nucleic acid samples, derived from Caco2 cell line DNA; The DNA from the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) was mixed with 4 mutant template DNAs in a certain proportion, and the mutation rates of the mixtures were 10%, 5%, 1%, and 0.1% respectively; among them, EGFR gene 19del mutation The DNA of the spot was derived from the H1650 cell line, the DNA containing the EGFR gene T790M mutation site was derived from the H1975 cell line (purchased from the Shanghai Institute of Biological Sciences, Chinese Academy of Sciences), and the DNA containing the EGFR gene L858R mutation site was derived from the H1975 cell line, The DNA containing the EGFR gene C797S mutation site is derived from a...

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Abstract

The invention provides a kit and method for detecting the mutation of an EGFR gene and particularly discloses a method, a primer and a probe for detecting the mutation of the EGFR gene, a kit including prime probe mixing liquid, a primer and a probe for detecting the mutation of a C797S locus, a primer and a probe for detecting the mutation of a T790M locus, a primer and a probe for detecting themutation of a 19del locus and a primer and a probe for detecting the mutation of a L858 locus. According to the method provided by the invention, 0.1% mutation rate can be detected based on a digitalPCR platform; and the method has the advantages that the optimization process is simple, much detected mutation types can be detected, the absolute quantification is realized, the sensitivity is high,samples are easily acquired, and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology and tumor diagnosis, in particular, the invention relates to a kit and a method for detecting EGFR gene mutation of non-small cell lung cancer. Background technique [0002] The morbidity and mortality of lung cancer rank first in the world, among which non-small cell lung cancer (NSCLC) accounts for more than 85%, and most patients are already in the advanced stage of cancer when they are diagnosed, and their mortality rate is as high as 80%-90%. Therefore, more and more people pay attention to the treatment of advanced NSCLC. Platinum-containing double-drug chemotherapy is still the main method for the treatment of advanced NSCLC, but the therapeutic effect is not very satisfactory. With the development of tumor molecular biology technology, the effect of drug-targeted therapy is more significant. Compared with traditional cytotoxic drugs, targeted therapy can act on the tumor site more precisely, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q1/6886C12Q2531/113C12Q2563/107
Inventor 蒋析文刘悦朱小亚颜尔聪
Owner DAAN GENE CO LTD
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