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Detection kit for lung cancer driving gene mutation

A detection kit and driving technology, which is applied in the field of detection kits for driving gene mutations in lung cancer, and can solve problems such as the inability to meet the actual needs of driving mutation gene detection

Active Publication Date: 2013-10-16
AMOY DIAGNOSTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have found that the amount of driver mutations is often lower than the detection limit that can be achieved by techniques such as direct sequencing, so techniques such as direct sequencing cannot meet the actual needs of gene detection for driver mutations

Method used

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  • Detection kit for lung cancer driving gene mutation
  • Detection kit for lung cancer driving gene mutation
  • Detection kit for lung cancer driving gene mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0185] This example takes the E746_T751del driver mutation on exon 19 of the EGFR gene, the Gly12Asp driver mutation of the KAS gene exon 12, the BRAF gene V600E driver mutation and the driver mutation of the EML4-ALK fusion gene Variant 1 as examples to illustrate the present invention. Fluorescence PCR was used to detect the driver mutations of the above genes.

[0186] One plasmid template strain (containing E746_T751del, Gly12Asp, V600E, Variant 1, 3a / b, 2 mutations) and 4 cell lines were used for the experiment, H460 (EGFR, KRAS and EM4-ALK gene wild type), 293T (EGFR, KRAS and EM4-ALK gene wild type), SW480 (EGFR, KRAS and EM4-ALK gene wild type), HT-29 (BRAF gene wild type), and 68 clinical lung cancer samples to be tested (including fresh tissue, paraffin section, pleural effusion, whole blood).

[0187] The method for detecting the E746_T751del driver mutation on exon 19 of the EGFR gene, the Gly12Asp driver mutation of the KAS gene exon 12, the driver mutation of th...

Embodiment 2

[0227] One case of paraffin-embedded tissue samples of clinical lung cancer sent to our company for testing in July 2011 was tested for driving mutations of EGFR, KRAS, BRAF and EML4-ALK.

[0228] Extraction of test sample DNA: Qiagen paraffin tissue sample extraction kit was used for DNA extraction, and the operation was performed according to the instructions of the extraction kit. The extracted DNA was dissolved in Tris-HCL (10 mmol / L, pH 8.0). The quality of the extraction was detected by a UV spectrophotometer to determine its concentration OD 260 / OD 280 1.9, then adjust the DNA concentration to 2ng / μL with Tris-HCL (10mmol / L, pH 8.0) solution as a PCR template. The DNA extracted above was used as templates for fluorescent PCR amplification of EGFR, KRAS, BRAF and EML4-ALK.

[0229] Sample mRNA was extracted using Qiagen (RNeasy FFPE kit) paraffin tissue sample extraction kit, and the operation was performed according to the instructions of the extraction kit. The ex...

Embodiment 3

[0258] Take 1 paraffin-embedded tissue sample from a lung cancer patient who was sent to our company in July 2011. EGFR, KRAS, BRAF, and EML4-ALK driver mutations were detected.

[0259] According to the detection kit and method described in Example 1, DNA and mRNA were extracted from the tissue samples, and the extraction steps were performed according to the operation steps of the kit instructions. Then use Tris-HCL (10mmol / L, PH 8.0) solution to adjust the DNA concentration to 2ng / μL as a PCR template. The template DNA was used as a template for fluorescent PCR amplification of EGFR, KRAS, BRAF and EML4-ALK.

[0260] The fluorescent PCR amplification system and amplification conditions of EGFR, KRAS, BRAF and EML4-ALK were operated according to the conditions described in Example 2, respectively. The MX3000P real-time PCR amplification instrument was used for amplification, and the fluorescence signals of FAM and HEX were detected after 35 cycles. Detect the Ct value of ...

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Abstract

The present invention discloses a detection kit for lung cancer driving gene mutation. The kit comprises: primers for detecting mutations of lung cancer driving genes including EGFR, KRAS, BRAF and EML4-ALK, and probes for detecting the mutations of the lung cancer driving genes including EGFR, KRAS, BRAF and EML4-ALK. The method of the present invention comprises: (1) providing 4 groups of the 71 primers and the probes; (2) extracting a DNA template of a sample requiring detection; (3) preparing a fluorescent PCR reaction system for detecting the driven mutations of EGFR, KRAS, BRAF and EML4-ALK; (4) adopting the hybridization of the dual-ring probe and the specific primer to detect the fluorescence intensity of FAM and HEX, or ROX of the reaction system, and determining the result according to the fluorescence intensity of the FAM and the HEX, or the ROX. With the method of the present invention, the driven mutations of the EGFR gene, the KRAS gene, the BRAF gene and the EML4-ALK gene of the lung cancer can be detected, and the method has characteristics of high sensitivity, strong specificity, rapid detection speed, convenient detection and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a detection kit for lung cancer driving gene mutation. Background technique [0002] Lung cancer is a common cancer worldwide, and its morbidity and mortality rank first among all cancers. In 2002, the new incidence rate of lung cancer in the world was 1.38 million, and nearly half (49.9%) occurred in developing countries. According to statistics from the Ministry of Health of my country in 2008, the mortality rate of lung cancer was 30.83 / 100,000. Lung cancer has become the malignant tumor with the highest morbidity and mortality, and the number of deaths from lung cancer is far greater than the sum of deaths from breast cancer, colon cancer, and prostate cancer (Orras JM, Fernandez E, Gonzalez JR, et., al. Lung cancer mortality in European regions (1955-1997). Ann Oncol, 2003, 14(1): 159-161; Ministry of Health of the People's Republic of China, "China Health Statistics Yearbook ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 阮力张海龙肖桃英施伟杰郑立谋
Owner AMOY DIAGNOSTICS CO LTD
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