Non-small cell lung cancer targeted therapy gene detection method

A technology for non-small cell lung cancer and targeted therapy, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve problems such as low throughput, difficult interpretation of results, poor repeatability, etc.

Inactive Publication Date: 2016-09-28
HEFEI INSTITUTES OF PHYSICAL SCIENCE - CHINESE ACAD OF SCI
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  • Abstract
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Problems solved by technology

[0011] In view of the existing non-small cell lung cancer detection methods that have problems such as difficult interpretation of results, poor repeatability, many false positives and false negatives, and low throughput, the present invention provides a non-small cell lung cancer targeted therapy gene detection method, which is a A method was developed based on multiplex PCR and high-throughput sequencing technology to detect 466 mutations in 12 oncogenes: AKT1, ALK, BRAF, EGFR, ERBB4, FGFR1, FGFR2, FGFR3, KRAS , MET, PIK3CA and PTEN, the mutations can be substitutions, insertions and / or deletions of one or more bases

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Embodiment 1

[0067] The sample used was fresh biopsy tissue of non-small cell lung cancer, and the genomic DNA in the fresh biopsy tissue of lung cancer was extracted with a DNA extraction kit, and the extracted genomic DNA was amplified by multiplex PCR using SEQ No. Illumina NextSeq 500 sequencer for sequencing analysis, the specific implementation method is as follows:

[0068] A. Genomic DNA extraction: Use the Qiagen DNeasy Blood&Tissue Kit to extract genomic DNA according to the kit instructions. The obtained genomic DNA is subjected to gel electrophoresis and quality detection using NanoDrop 2000. It is required that the genomic DNA is not significantly degraded, the concentration is greater than 20ng / μL, A260 / A280>1.8, A260 / A230>2.0, and Agilent Bioanalyzer 2100 is used for accurate quantification .

[0069]B. Multiplex PCR amplification: Use SEQ No.1 to SEQ No.96 to perform multiplex PCR amplification on 10ng of genomic DNA to obtain AKT1, ALK, BRAF, EGFR, ERBB4, FGFR1, FGFR2, FG...

Embodiment 2

[0082] Using primers from SEQ No.1 to SEQ No.96, multiplex PCR amplification was performed on the genomic DNA extracted from paraffin-embedded non-small cell lung cancer tissues. Sequencing analysis, the specific implementation method is as follows:

[0083] A. Genomic DNA extraction: Use the Invitrogen MagMAX FFPE DNA Isolation Kit to extract the genomic DNA from paraffin-embedded tissues according to the manufacturer's recommended operating procedures. The quality of the obtained genomic DNA was tested using a Qubit 3.0 fluorometer, and the concentration was required to be greater than 20ng / μL, A260 / A280>1.8, and A260 / A230>2.0. Included on the genomic DNA are those required for the present invention.

[0084] B. Multiplex PCR amplification: 466 mutation sites contained in the 12 target genes of AKT1, ALK, BRAF, EGFR, ERBB4, FGFR1, FGFR2, FGFR3, KRAS, MET, PIK3CA and PTEN on the genomic DNA in step A The region was amplified by multiplex PCR using primers SEQ No.1 to SEQ No...

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Abstract

The invention discloses a non-small cell lung cancer targeted therapy gene detection method, and belongs to the field of gene detection. The method for detecting 466 mutations of 12 oncogenes is developed by multiplex PCR and high throughput sequencing technologies, wherein the oncogenes are AKT1, ALK, BRAF, EGFR, ERBB4, FGFR1, FGFR2, FGFR3 , KRAS, MET, PIK3CA, and PTEN, and the mutations may be substitutions, insertions and / or deletions of one or more bases. The detection method provided by the invention has the advantages of high detection sensitivity of up to 0.01%, and clear and objective detection results, can be directly used for reflecting specific mutation sites of the relevant gene, directly used for guiding clinical non-small cell lung cancer targeted dosage, and used for early diagnosis or auxiliary diagnosis and screening of cancers as well as post-cancer surveillance.

Description

technical field [0001] The invention belongs to the field of gene detection, and more specifically relates to a gene detection method for non-small cell lung cancer targeted therapy. Background technique [0002] The third national survey on the causes of population death showed that in the past 30 years, the mortality rate of lung cancer in my country has increased by 465%, surpassing liver cancer to become the cancer with the highest mortality rate. It is estimated that in 2015, there were 733,300 new cases of lung cancer in my country (509,300 males and 224,000 females), and 610,200 deaths (432,400 males and 177,800 females), ranking first among all malignant tumors in terms of morbidity and mortality. . Therefore, lung cancer has become a very important public health problem in our country. [0003] Lung cancer can be divided into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) according to histology. Non-small cell lung cancer accounts for about 80...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/686C12Q1/6806C12Q1/6869C12Q2537/143C12Q2525/191C12Q2535/122C12Q2563/143C12Q2563/149
Inventor 林文楚王伟洪波李红高小平王伙刚
Owner HEFEI INSTITUTES OF PHYSICAL SCIENCE - CHINESE ACAD OF SCI
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