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Kit for quantitatively detecting EGFR mutation

A kit and chain reaction technology, which is applied in the determination/testing of microorganisms, DNA/RNA fragments, and the introduction of foreign genetic material using vectors, etc., which can solve problems such as inability to quantify

Inactive Publication Date: 2011-04-20
BEIJING ACCB BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Other methods, such as allele-specific oligonucleotide probe hybridization, are very sensitive to hybridization conditions and require strict control of experimental conditions; restriction fragment length polymorphism experiments require considerable manpower and cannot be quantified

Method used

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  • Kit for quantitatively detecting EGFR mutation
  • Kit for quantitatively detecting EGFR mutation
  • Kit for quantitatively detecting EGFR mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Human fresh tumor tissue, paraffin-embedded tissue, peripheral blood, pleural effusion, human Genomic DNA Extraction from Cell Lines

[0030] The human cancer cell lines we tested include non-small cell lung cancer (NSCLC) (A549, H460, H838 and H1703), breast cancer (MCF-7, BT474 and HuL100), malignant multiple mesothelioma cell lines (H513, H2052, H290, MS-1 and H28), colon cancer cell lines (SW480), head and neck cancer cell lines (U87), cervical cancer (Hela), sarcoma cell lines (Mes-SA, Saos-2 and A204).

[0031] Human fresh tumor tissues, peripheral blood, and paraffin-embedded tissues that we tested included NSCLC, mesothelioma, colon cancer, malignant melanoma, renal cancer, esophageal cancer, thyroid cancer, malignant tumor, and ovarian cancer.

[0032] Specimen DNA Extraction

[0033] Can use the DNA extraction kit of Qiagen Company, Promega Company, Roche Company to extract sample genomic DNA, use Gene Company Nanodrop ND1000 type nucleic acid ...

Embodiment 2

[0088] Example 2: Preparation of plasmid standards containing mutant and wild-type detection sequences

[0089] 1. Wild-type plasmid construction ( figure 1 ， figure 2 )

[0090] 1.1 Preparation of the carrier

[0091] The TA cloning vector pMD18-T was purchased from TAKARA Company.

[0092] 1.2 Preparation of insert fragments

[0093] Use the PCR method to prepare the insert fragment. The template for PCR is the sample genomic DNA extracted in step 1. The reaction system and amplification conditions are as follows (Table 1, Table 2, Table 3):

[0094] Table 1: PCR reaction system (50μl)

[0095]

[0096] To prepare a plasmid containing the wild-type sequence at position 2155 of exon 18 of the EGFR gene, E18-F-1 (SEQ ID NO: 5) or E18-F-2 (SEQ ID NO: 6) and E18-R-1 (SEQ ID NO: 7) or E18-R-2 (SEQ ID NO: 8) primer sequence; if the preparation contains EGFR gene exon 19 2235-2249, 2236-2250, 2254- For the wild-type plasmid at position 2277, E19-F-1 (SEQ ID NO: 9) or E1...

Embodiment 3

[0116] Example 3: Taking lung cancer and cervical cancer samples as examples: human cell lines, human fresh tumors Detection of EGFR mutation in genomic DNA of tumor tissue, peripheral blood, and paraffin-embedded tissue

[0117] 1. The fluorescent quantitative PCR reaction template is the genomic DNA of lung cancer and cervical cancer samples extracted in Example 1 and the standard prepared in Example 2, and double distilled water is used as a negative control. In order to make a standard curve, standard dilutions were 1ng / μl, 0.5ng / μl, 0.25ng / μl, 0.125ng / μl, 0.0625ng / μl, 0.03125ng / μl.

[0118] 2. Reaction system and reaction conditions (table 2, table 5, table 6, table 7), wherein labeling probe fluorescent emission group is selected from: FAM, TET, HEX, ROX; Fluorescence quencher group is selected from: BHQ, TAMARA.

[0119] Table 5: Real-time quantitative PCR reaction system (20μl / tube)

[0120]

[0121] To detect the G→A mutation at position 2155 of exon 18 of th...

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Abstract

The invention relates to an epidermal growth factor receptor (EGFR) gene mutation detection method and a detection kit related with molecular targeted anti-cancer medicament curative effect, in particular to a fluorescent quantitative polymerase chain reaction (PCR) detection method for detecting mutation in an EGFR gene mutation hotspot area, a kit and application thereof. The method and the kit detect the mutation of specific sites of EGFR genes, and can predict the curative effect of a molecular targeted anti-cancer medicament EGFR tyrosine kinase inhibitor so as to provide guidance for clinical individual administration schemes of tumor patients.

Description

Background of the invention [0001] Epidermal growth factor receptor (EGFR) is a multifunctional glycoprotein widely distributed on the cell membrane of various tissues in the human body. It is a member of the HER / ErbB family and is associated with tumor proliferation, angiogenesis, tumor metastasis and related to apoptosis. [0002] Most tumor cells express EGFR and its natural ligands, which can cause their own phosphorylation after binding, and transmit the signal to the nucleus through a series of reactions, thereby affecting the growth and apoptosis of tumor cells and tumor angiogenesis to affect tumor growth. development and evolution. [0003] Studies have shown that EGFR plays an important role in the initiation and progression of cancer. Targeted therapy drugs targeting EGFR, such as Avastin (AVASTIN), Tarceva (Erlotinib), Gefitinib (Irressa) and other EGFR tyrosine kinase inhibitors (EGFR-TKI) can block tumor cell EGFR Signal transduction to inhibit tumor cell prol...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C12N15/63
CPCC12Q1/6851C12Q2600/156C12Q1/6827C12Q1/6886
Inventor 许军普陈钊李隽
Owner BEIJING ACCB BIOTECH
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