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Primers and probes for detecting human EGFR gene mutations as well as use method thereof

A probe and human technology, applied in the fields of biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve problems such as the inability to meet the needs of non-invasive mutation detection, and achieve fast detection speed and selectivity. Strong, specific effect

Active Publication Date: 2011-06-29
AMOY DIAGNOSTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above methods cannot meet the increasing needs of non-invasive mutation detection, so the ability to accurately and non-invasively detect such rare mutations poses a challenge to existing detection technologies

Method used

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  • Primers and probes for detecting human EGFR gene mutations as well as use method thereof
  • Primers and probes for detecting human EGFR gene mutations as well as use method thereof
  • Primers and probes for detecting human EGFR gene mutations as well as use method thereof

Examples

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Effect test

Embodiment 1

[0058] In this example, the E746_A750del (2235_2249del15) mutation on exon 19 of the EGFR gene hotspot mutation is taken as an example to analyze the method for detecting 19 deletion mutations of the EGFR gene by single-tube fluorescent PCR of the present invention. Four cell lines were used in the experiment, H1650 (with E746_A750del mutation), H460 (EGFR gene wild type), 293T (EGFR gene wild type), SW480 (EGFR gene wild type); 100 whole blood samples from healthy unpaid blood donors , 62 clinical lung cancer samples (including fresh tissue, paraffin section, pleural effusion, whole blood).

[0059] The method for detecting the E746_A750del (2235_2249del15) mutation on exon 19 of the EGFR gene hotspot mutation using the above-mentioned fluorescent PCR comprises the following steps:

[0060] (1) Sample processing and template extraction quality control:

[0061] The scope of application of samples includes surgically removed fresh pathological tissues, formaldehyde-fixed para...

Embodiment 2

[0093] In this example, the L747_P753>S(2240_2257del18) mutation on exon 19 of the EGFR gene hotspot mutation is taken as an example to analyze the method for detecting 19 deletion mutations of the EGFR gene by single-tube fluorescent PCR of the present invention. One plasmid template strain (including L747_P753>S mutation) was used in the experiment, three cell lines were H460 (EGFR gene wild type), 293T (EGFR gene wild type), SW480 (EGFR gene wild type); 100 healthy free Whole blood samples from blood donors, 62 clinical lung cancer samples (including fresh tissue, paraffin sections, pleural effusion, whole blood).

[0094] The method for detecting the L747_P753>S (2240_2257del18) mutation on exon 19 of the EGFR gene hotspot mutation by using the above fluorescent PCR includes the following steps:

[0095] (1) Sample processing and template extraction quality control:

[0096] The scope of application of samples includes surgically removed fresh pathological tissues, formal...

Embodiment 3

[0119] In this example, the E746_T751del (2236_2253del18) mutation on exon 19 of the EGFR gene hotspot mutation is taken as an example to analyze the method for detecting 19 deletion mutations of the EGFR gene by single-tube fluorescent PCR of the present invention. One plasmid template strain (including E746_T751del mutation) was used for the experiment, three cell lines were H460 (EGFR gene wild type), 293T (EGFR gene wild type), SW480 (EGFR gene wild type); 100 healthy voluntary blood donors Whole blood samples, 62 clinical lung cancer samples (including fresh tissue, paraffin sections, pleural effusion, whole blood).

[0120] The method for detecting the L747_P753>S (2240_2257del18) mutation on exon 19 of the EGFR gene hotspot mutation by using the above fluorescent PCR includes the following steps:

[0121] (1) Sample processing and template extraction quality control:

[0122] The scope of application of samples includes surgically removed fresh pathological tissues, fo...

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Abstract

The invention discloses primers and probes for detecting human EGFR gene mutations as well as a use method, relating to the detection of gene mutation. The method of the invention comprises the following steps: (1) providing the primers and the probes; (2) processing the sample to be detected and extracting a template; (3) preparing a reaction system of fluorescent PCR amplification mutation genesequence; (4) amplifying the mutable gene target sequence by the primers and the probes in the step (1); (5) detecting the fluorescence intensity of FAM and HEX or ROX of the reaction system as the judgment standard of results by adopting hybridization of a dual ring probes and an amplification product. The primers, the probes and the method of the invention can simultaneously detect 29 deletion mutations of EGFR gene, and features high sensitivity, strong specificity, strong selective capacity and fast detection speed demonstrated by completing the whole detection process in only 90 minutes.

Description

technical field [0001] The invention relates to gene mutation detection, in particular to primers, probes and methods for detecting 29 deletion mutations of human epidermal growth factor receptor EGFR gene. Background technique [0002] Human epidermal growth factor receptor (EGFR) exists in all normal epithelial and some mesenchymal cells, and plays an important role in the regulation of cell growth and differentiation. EGFR can activate tyrosine kinases, thereby opening downstream signaling pathways. Therefore, EGFR has become an important target in the research of targeted therapy for tumors, especially for non-small cell lung cancer. At present, clinical EGFR-targeted therapy mainly includes small molecule tyrosine kinase inhibitors and monoclonal antibodies, such as AstraZeneca’s gefitinib (Iressa), Roche’s erlotinib ( Troquet). [0003] In recent years, a large number of studies have found that some lung cancer patients have mutations in the coding region of the EGF...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11G01N21/64
Inventor 阮力何东华郑立谋
Owner AMOY DIAGNOSTICS CO LTD
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