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Kit and method for detecting gene mutation of EGFR gene exon 21

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

Inactive Publication Date: 2018-12-14
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 gene mutation of EGFR gene exon 21
  • Kit and method for detecting gene mutation of EGFR gene exon 21
  • Kit and method for detecting gene mutation of EGFR gene exon 21

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preparation example Construction

[0100] Step 2, preparation of dPCR system

[0101] 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. :

[0102] Table 5 dPCR system

[0103]

[0104]

[0105] The nucleotide sequence information of the primer probe is as follows:

[0106] Nucleotide sequence information of primers and probes for detection of L858R site mutation in exon 21:

[0107] Upstream primer (SEQ ID NO: 1) 5'-AAAACACCGCAGCATGTCAAG-3';

[0108] Downstream primer (SEQ ID NO: 2) 5'-GCCACCTCCTTACTTTGCCTC-3';

[0109] Mutation probe (SEQ ID NO: 3) 5'-FAM-TTTGGGCGGGCCAAACTG-MGB-3';

[0110] Wild probe (SEQ ID NO: 4) 5'-VIC-TTTTGGGCTGGCCAAACT-MGB-3'.

[0111] The probes include a mutation probe and a wild probe for detecting the L858...

Embodiment 1

[0132] This embodiment provides a test kit for detecting EGFR gene mutation in patients with non-small cell lung cancer, its composition, packaging and quantity (48 reactions / box), as shown in Table 7,

[0133] Table 7 The composition, packaging and quantity of the kit

[0134]

Embodiment 2

[0135] Embodiment 2: Sensitivity detection and mutation detection rate experiment

[0136]The gDNA control sample is a nucleic acid sample containing the wild-type EGFR gene L858R, which is derived from the DNA of a Caco2 cell line (purchased from the Shanghai Institute for Biological Sciences, Chinese Academy of Sciences); Proportionally mixed, the mutation rates of the mixture were 10%, 5%, 1%, and 0.1%, respectively; wherein, the DNA containing the EGFR gene L858R mutation site was derived from H1975 cell line (purchased from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences); Negative control is sterile water;

[0137] Take 5 μL each of the negative control, gDNA control, and sensitivity reference substance, and add the sample to the eight-tube tube of the dPCR reaction system prepared in step 2, so that the total volume of each tube of dPCR reaction solution is 16 μL; tightly cover the tube cap of the eight-tube tube, mix thoroughly Uniformly, high...

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Abstract

The invention provides a kit and a method for detecting gene mutation of EGFR gene exon 21, and concretely discloses a method, a primer and a probe for detecting mutation of a free nucleic acid EGFR gene in blood of a patient with non-small cell lung cancer, and a kit containing the primer and probe mixed solution. The method and the kit are based on a digital PCR platform, and have the advantagesof simple optimization process, multiple datable mutation types, absolute quantification, high sensitivity, and easiness in obtaining of a sample.

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 mutation of exon 21 of EGFR gene. 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, which can signif...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886C12Q2600/118C12Q2600/156
Inventor 蒋析文朱小亚刘悦颜尔聪
Owner DAAN GENE CO LTD
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