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Method and kit for detecting mutation of EGFR gene

A kit and gene technology, applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc., can solve the problems of long detection cycle, low detection sensitivity, complicated steps, etc., and improve the specificity of primers , Improving detection sensitivity and ensuring accuracy

Inactive Publication Date: 2017-07-07
杭州联川基因诊断技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The purpose of the present invention is to provide a detection method and kit for EGFR gene mutation based on multiplex PCR and high-throughput sequencing technology, aiming to solve the problems of complicated steps, low detection sensitivity and long detection cycle in the prior art

Method used

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  • Method and kit for detecting mutation of EGFR gene
  • Method and kit for detecting mutation of EGFR gene
  • Method and kit for detecting mutation of EGFR gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] The reliability of the kit is verified by detecting the standard of known mutation type and frequency.

[0039] A. FFPE DNA extraction

[0040] Use the extraction kit QIAamp DNA FFPE Tissue Kit (Qiagen, Cat. No. 56404) to extract FFPE DNA from a piece of paraffin coil (Horizon, Cat. No. HD200), and the operation is as described in the kit instruction manual. The resulting product is used Fluorescence Quantitative Instrument and Corresponding The quantitative reagent (Invitrogen) was used for quantification, and 350 ng of total FFPEDNA was obtained.

[0041] B. multiplex PCR method to amplify the target region of the genome of the sample to be tested.

[0042] 1). Prepare a sterile, nuclease-free 200 μL PCR tube and place it on ice; prepare the PCR reaction system as shown in the table below, and be careful to avoid cross-contamination.

[0043]

[0044] The basic information of the primers is as follows, N means A, C, G or T:

[0045]

[0046]

[0047] 2...

Embodiment 2

[0069] The reliability of this kit is verified by comparing the mutation type and frequency of the patient's plasma cell-free DNA or FFPE DNA with the results of digital PCR (ddPCR).

[0070] A. Cell-free DNA extraction from patient plasma

[0071] Use the extraction kit QIAamp Circulating Nucleic Acid Kit (Qiagen, product number 55114) to extract cfDNA from the plasma of the 3 patients, and the operation is as described in the extraction kit instructions; use the extraction kit QIAampDNA FFPE Tissue Kit (Qiagen, product number 56404) The patient's FFPE tissue samples were extracted according to the instructions of the extraction kit. The resulting product is used Fluorescence Quantitative Instrument and Corresponding Quantification reagent (Invitrogen) was used for quantification.

[0072] B. multiplex PCR method to amplify the target region of the genome of the sample to be tested.

[0073] 1). Prepare a sterile, nuclease-free 200 μL PCR tube and place it on ice; prepa...

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Abstract

The invention discloses a method and a kit for detecting mutation of an EGFR gene. The method comprises the steps of bonding a specific primer with a target template DNA sequence, amplifying a to-be-tested sample target region by virtue of a universal primer, purifying a library by virtue of magnetic beads, carrying out high-throughput sequencing on the obtained library, and analyzing the mutation of the EGFR gene. The kit comprises the primers, a PCR mixing reagent, a positive control and the magnetic beads. By utilizing the method and the kit, the experimental steps can be simplified, and the detection sensitivity is improved. The method and the kit are applicable to the rapid detection of the mutation of the EGFR gene of clinic tumor patients and are helpful for guiding clinic personalized medication.

Description

technical field [0001] The invention relates to the field of gene mutation diagnosis of molecular biology, and more specifically relates to a method and a kit for detecting EGFR gene mutation. Background technique [0002] In recent years, the technology of using molecular targeted drugs to treat tumors has developed rapidly. This method is different from traditional chemotherapy. It uses targeted drugs to bind to the characteristic sites of tumor cells to intervene in the gene signal transduction pathway that controls the growth and proliferation of tumor cells; Can cause damage to normal cells, which has a huge advantage. [0003] Human epidermal growth factor receptor (Epidermal Growth Factor Receptor, EGFR) is a transmembrane glycoprotein with a molecular weight of 170kD composed of 1186 amino acids. It is mainly located on the cell membrane and widely expressed in most normal epithelial cells. Its structure includes cell The outer binding region, the transmembrane regi...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/6886C12Q2600/156C12Q2600/16C12Q2537/143C12Q2535/122C12Q2563/185
Inventor 郎秋蕾周小川李璐璐方超梁洪林彬张瑞静
Owner 杭州联川基因诊断技术有限公司
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