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Lung cancer gene spectrum detection method and kit based on Mass ARRAY platform Iplex analysis and application of kit

A detection kit and detection method technology, applied in the detection field of lung cancer detection, can solve the problems of high price and the like

Active Publication Date: 2015-09-09
GUANGDONG GENERAL HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because the kit is developed abroad, the technical information is confidential, the price is expensive, and it does not fully include cutting-edge molecular targets such as primary MET gene mutations related to EGFR TKI resistance to targeted therapy, multiple site mutations in the ALK gene and Crizotinib is related to targeted therapy drug resistance, etc., and the driving gene profiles in China and the United States are different. Therefore, it is of great significance to develop a multi-gene simultaneous detection method suitable for the Chinese population.

Method used

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  • Lung cancer gene spectrum detection method and kit based on Mass ARRAY platform Iplex analysis and application of kit
  • Lung cancer gene spectrum detection method and kit based on Mass ARRAY platform Iplex analysis and application of kit
  • Lung cancer gene spectrum detection method and kit based on Mass ARRAY platform Iplex analysis and application of kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0103] Example 1 Preparation of Lung Cancer Multigene Spectrum Detection Primer Kit

[0104] 1. Determine lung cancer-related driving genes or targeted drug resistance genes: Search the PUBMED database through the Internet, consult the latest literature, and count the targeted genes or their parallels related to the pathogenesis, chemoradiotherapy, targeted drug resistance, and metastasis of lung cancer in the Chinese population. , adjacent pathway genes, the genes (13) determined to be included in the kit are as follows: EGFR (NM_005228), KRAS (NM_033360), ALK (NM_004304), FGFR1 (NM_023110.2), FGFR2 (NM_000141.2), FGFR3 (NM_000142 .4), PIK3CA(NM_006218.2), BRAF(NM_004333.4), PTEN(NM_000314.4), MET(NM_001127500.1), ERBB2(NM_004448), AKT1(NM_005163), STK11(NM_000455.4).

[0105] 2. Screen the hotspot mutation sites of the target gene: In the COSMIC database, the COSMIC gene numbers of the above 13 genes are EGFR (ENST00000275493), KRAS (ENST00000256078), ALK (ENST00000389048), ...

Embodiment 2

[0111] Example 2 Detection of lung cancer cell lines using the kit of Example 1

[0112] 1. Select 8 lung cancer cell lines (all of which can be purchased from ATCC) with known mutation sites H460, PC9, H1650, H1975, A549, GLC82, HCC827, and H1299 to carry out the feasibility analysis of this kit, and set up normal Human genomic DNA (gDNA) (derived from the foreskin tissue extraction of normal people in Guangdong Provincial People's Hospital) was used as a negative control, and water (H 2 O) as a blank control. The detection of each sample requires 120ng of DNA, that is, 10ng / well×12 wells. The operation of the kit of the present invention is as follows:

[0113] (1) The PCR system is: 0.5 μl of 10×PCR buffer, MgCl 2(25mM) 0.4μl, dNTPs (25mM) 0.1μl, PCR enzyme (5U / μl) 0.2μl, amplification primer mixture 1μl, lung cancer tissue sample genome (10ng / μl) 2μl, make up to 5μl with water. 94°C for 2 minutes; 94°C for 30 seconds, 56°C for 30 seconds, 72°C for 1 minute, 45 cycles; ...

Embodiment 3

[0121] Example 3 Using the kit of Example 1 to detect lung cancer patient tissue samples

[0122] 1. For verification, 3 human lung adenocarcinoma tissue samples (from Guangdong Provincial People's Hospital) with mutations detected by the LungCarta kit and 3 lung cancer tissue samples (from Guangdong Provincial People's Hospital) with no mutation detected were selected. Human genomic DNA (gDNA) was used as a negative control, water (H 2 O) as a blank control. The detection of each sample requires 120ng of DNA, that is, 10ng / well×12 wells. Wherein, the LungCarta kit was operated according to its instructions. The operation of the kit of the present invention is as follows:

[0123] (1) The PCR system is: 0.5 μl of 10×PCR buffer, MgCl 2 (25mM) 0.4μl, dNTPs (25mM) 0.1μl, PCR enzyme (5U / μl) 0.2μl, amplification primer mixture 1μl, lung cancer tissue sample genome (10ng / μl) 2μl, make up to 5μl with water. 94°C for 2 minutes; 94°C for 30 seconds, 56°C for 30 seconds, 72°C for 1...

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Abstract

The invention discloses a lung cancer gene spectrum detection method and kit based on Mass ARRAY platform Iplex analysis and application of the kit. The method can be used for detecting 99 hot point mutation loci of 13 driving and drug resistance related genes of lung cancer; amplification primers shown in SEQ ID NO.1-198 are used for amplification reaction and then for digestion reaction; extension primers shown in SEQ ID NO.199-297 are used for extension reaction to obtain a product; and the product is subjected to Mass ARRAY platform Iplex analysis. A detection kit designed according to the method keeps up with the international frontier of lung cancer diagnosis and treatment and takes the difference between gene spectrums of European and American people and Asians into consideration and has more detection loci; moreover, the detection kit has the advantages of low price, high throughput, shot time consumption, no limit to detection samples, high accuracy and high sensitivity. Therefore, the kit has a huge potential in clinical application.

Description

technical field [0001] The invention relates to a detection method for detecting lung cancer, in particular to a lung cancer gene spectrum detection method based on MassARRAY mass spectrometry platform Iplex analysis, a kit and an application. Background technique [0002] Lung cancer is the first cause of death from malignant tumors in China. The incidence rate continues to rise and the health hazard situation is severe. Its occurrence, development and treatment are closely related to gene mutations. The "individualized" targeted therapy based on gene mutations is the next step after traditional radiotherapy and chemotherapy. new breakthroughs in the field. Gene target mutations in non-small cell lung cancer (NSCLC) samples can benefit from corresponding targeted drug therapy, the tyrosine kinase inhibitor (TKI) gefitinib / erine for EGFR and ALK gene mutations Targeted therapies such as erlotinib and crizotinib can prolong survival for patients. With the deepening of resea...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6827C12Q1/6886C12Q2600/156C12Q2565/501C12Q2565/627
Inventor 吴一龙张绪超田红霞
Owner GUANGDONG GENERAL HOSPITAL
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