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Kit for detecting lung cancer EGFR gene mutation targeted therapy related drug-resistant variation sites

A technology of targeted therapy and mutation sites, applied in the field of clinical biology, to optimize economic benefits, reduce clinical testing costs, and achieve throughput effects

Inactive Publication Date: 2017-03-29
GUANGDONG GENERAL HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are few reports at home and abroad on the kits for the simultaneous detection of EGFR-targeted therapy drug resistance genes

Method used

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  • Kit for detecting lung cancer EGFR gene mutation targeted therapy related drug-resistant variation sites
  • Kit for detecting lung cancer EGFR gene mutation targeted therapy related drug-resistant variation sites
  • Kit for detecting lung cancer EGFR gene mutation targeted therapy related drug-resistant variation sites

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1 Selection of Drug-Resistant Mutation Sites Related to EGFR Gene Mutation Targeted Therapy in Lung Cancer

[0059] According to the latest research progress of lung cancer EGFR gene mutation targeted therapy, the description and frequency distribution of mutation sites in the COSMIC database, and the previous research basis of this lung cancer research institute, the present invention incorporates the detection of lung cancer EGFR gene mutation targeted therapy drug resistance. 60 base variation sites of 7 genes. These genes and loci are from the loss of sensitive mutations in EGFR gene leading to drug resistance (such as Exon19 deletion, loss of sensitive mutations such as L858R, G719X, L861Q); drug resistance loci of secondary mutations in EGFR gene (p. , p.C797YSA, p.L747S, p.D761YN, p.T854APS, Exon20 insertion mutations); activation of bypass pathways or downstream pathways (related drug resistance mutation sites of KRAS, BIM, PIK3CA, BRAF, NRAS genes) thre...

Embodiment 2

[0063] Example 2 Preparation of primer kits for drug resistance mutation sites related to lung cancer EGFR gene mutation targeting therapy

[0064] 1. Software design multiple primer documents: According to Example 1, it is determined that the 7 genes included in the kit are EGFR (NM_005228), KRAS (NM_033360), PIK3CA (NM_006218.2), BRAF (NM_004333.4), ERBB2 (NM_004448), BIM (NM_138621.4), NRAS (NM_002524.4). The gene numbers of the above seven genes in the COSMIC database are EGFR (ENST00000275493), KRAS (ENST00000256078), PIK3CA (ENST00000263967), BRAF (ENST00000288602), ERBB2 (ENST00000269571), BIM (ENST_00000393250)3. In the PUBMED database, the genomic gDNA sequences of the 7 genes included in the kit were EGFR (NG_007726.3), KRAS (NG_007524.2), PIK3C (NG_012113.2), BRAF (NG_007873.3), ERBB2 (NG_007503.1 ), BIM (NG_029006.1), NRAS (NG_007572.1). According to the CDS sequence of the gene in the COSMIC database, 60 variant sites of 7 genes were marked in the gDNA sequence,...

Embodiment 3

[0078] Example 3 Detection of lung cancer cell lines using the drug resistance kit of Example 2

[0079] 1. Select nine lung cancer cell lines (all of which can be purchased from ATCC) with known mutation sites, H460, PC9, H1650, H1975, A549, GLC82, HCC827, H2228, and H1299, to carry out the feasibility analysis of this kit. Set up normal human genome DNA (gDNA) (Promega company human genome gDNA: Male as 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. The operation of the kit of the present invention is as follows:

[0080] (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; 72°C for 5 minutes;

[0081] (2) Add 0.3 μl of exonucl...

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Abstract

The invention discloses a kit for detecting lung cancer EGFR gene mutation targeted therapy related drug-resistant variation sites. The kit comprises amplimers represented by SEQ ID NO.1 to SEQ ID NO.122 and extension primers represented by SEQ ID NO.123 to SEQ ID NO.183. The kit can detect 60 nucleic acid sites of the following seven relevant genes: EGFR, KRAS, PIK3CA, BRAF, HER2, BIM and NRAS, provides gene analysis data for successful or failed EGFR targeted therapy, and provides basis for subsequent therapy and researches.

Description

technical field [0001] The invention belongs to the field of clinical biotechnology, and in particular relates to a kit for detecting drug-resistant mutation sites related to lung cancer EGFR gene mutation targeted therapy. Background technique [0002] Individualized therapy with targeted drugs has brought a new dawn to patients with non-small cell lung cancer (NSCLC), but most lung cancer patients with genetic mutations eventually become resistant to tyrosine kinase inhibitors (TKIs) about one year after receiving treatment. The molecular mechanism by which resistance emerges to a drug is usually one of two types: (1) mutation or gene copy number gain affecting the primary target; (2) changes in other genes or gene products within the target or in parallel signaling pathways , to compensate or bypass target signal inhibition. [0003] The molecular mechanism of non-small cell lung cancer resistance to EGFR TKI drugs erlotinib / gefitinib mainly lies in EGFR-T790M mutation, ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6858C12Q1/6886C12Q2600/106C12Q2600/156C12Q2600/16C12Q2537/143C12Q2531/113C12Q2565/627
Inventor 吴一龙田红霞张绪超杨衿记王震郭伟浜
Owner GUANGDONG GENERAL HOSPITAL
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