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Method for detecting EGFR (epidermal growth factor receptor) mutant gene based on multiple-fluorescence PCR (polymerase chain reaction)

A mutant gene and multiple fluorescence technology, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems of limitation, low sensitivity and detection throughput, and it is difficult to realize one-time simultaneous detection of EGFR mutant genes, etc., to achieve The effect of strong specificity and high accuracy

Pending Publication Date: 2019-03-12
SHANGHAI MAG GENE NANOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the ARMS-PCR method is a relatively simple and highly sensitive detection method, it is difficult to realize the simultaneous detection of EGFR mutation genes due to the limitation of the PCR detection channel.
[0009] Therefore, in order to simultaneously solve the problems of low sensitivity and detection throughput faced by the existing EGFR mutation gene detection technology, the present invention is committed to developing a new EGFR mutation site detection method and kit

Method used

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  • Method for detecting EGFR (epidermal growth factor receptor) mutant gene based on multiple-fluorescence PCR (polymerase chain reaction)
  • Method for detecting EGFR (epidermal growth factor receptor) mutant gene based on multiple-fluorescence PCR (polymerase chain reaction)
  • Method for detecting EGFR (epidermal growth factor receptor) mutant gene based on multiple-fluorescence PCR (polymerase chain reaction)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: (Detection of E746-A750del / EX20ins on exon 19 of EGFR gene hotspot mutation)

[0041] 1. Extract template DNA:

[0042] Template DNA was extracted from patient tissue samples (including fresh tissue, paraffin sections, pleural effusion, and whole blood) using other commercially available kits. In addition, 10,000 copies of wild template (A549) and mutant gene (Del 19 / 20insert) in different proportions were added to the reaction solution, and then amplified and detected by the present invention to verify its reliability.

[0043] 2. PCR amplification

[0044] According to the positional relationship of the EGFR mutation types to be tested in this project, we designed 7 pairs of primers to amplify different mutation types of EX19DEL / EX20ins, and the amplification products of SEQ ID NO.8 and SEQ ID NO.9 contain EX19DEL mutation types ; The amplification products of SEQ ID NO.15 and SEQ ID NO.16 / SEQ ID NO.17 / SEQ ID NO.18 / SEQ ID NO.19 / SEQ ID NO.20 / SEQ ID NO.21 c...

Embodiment 2

[0053] Example 2: Mutation detection of EGFR gene hotspot mutation exon T790M / L858R

[0054] 1. Extract template DNA:

[0055]Template DNA was extracted from patient tissue samples (including fresh tissue, paraffin sections, pleural effusion, and whole blood) using other commercially available kits. In addition, 10,000 copies of wild template (A549) and different ratios of mutant genes (T790M / L858R) were added to the reaction solution, and then amplified and detected by the present invention to verify its sensitivity.

[0056] 2. PCR amplification

[0057] According to the positional relationship of the EGFR mutation types to be tested in this project, we designed 2 pairs of primers to amplify different mutation types of L858R / T790M, and the amplification products of SEQ ID NO.22 and SEQ ID NO.23 contain the L858R mutation type ; The amplified products of SEQ ID NO.24 and SEQ ID NO.25 contain T790M mutation type.

[0058] Synthesis of PCR primers / specific probes: The synthe...

Embodiment 3

[0067] 1. Extract template DNA:

[0068] Template DNA was extracted from patient tissue samples (including fresh tissue, paraffin sections, pleural effusion, and whole blood) using other commercially available kits. In addition, 10,000 copies of wild template (A549) and different proportions of mutant genes (G719X / S768I / L861Q) were added to the reaction solution, and then amplified and detected by the present invention to verify its sensitivity.

[0069] 2. PCR amplification

[0070] According to the positional relationship of the EGFR mutation types to be tested in this project, we designed 6 pairs of primers (please correct) to amplify different mutation types of G719X / S768I / L861Q, SEQ ID NO.10 and SEQ ID NO.11 / SEQ The amplification product of IDNO.12 / SEQ ID NO.13 / SEQ ID NO.14 comprises G719X mutation type; The amplification product of SEQ ID NO.26 and SEQID NO.27 comprises S768I mutation type; SEQ ID NO.28 and SEQ ID NO.28 and SEQ ID NO.27 The amplified product of ID NO.2...

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Abstract

The invention discloses a method for detecting EGFR (epidermal growth factor receptor) mutant gene based on multiple fluorescence, relates to the field of detection of the EGFR mutant gene, and provides a method for detecting the EGFR mutant gene based on the multiple-fluorescence PCR (polymerase chain reaction). The method comprises the following steps of preparing multiple specific ARMS (amplification refractory mutation system)-amplification primers for different EGFR mutant genes and blockers for inhibiting amplification of wild genes, amplifying a fragment containing the mutant gene in ato-be-detected sample, and judging whether the different samples have the corresponding EGFR gene mutation or not according to the fluorescence value. The invention also provides a kit for detecting the EGFR mutant gene through the method. The method has the advantages that the sensitivity is high, the accuracy is high, and the specificity is strong; the multiple EGFR mutant genes can be simultaneously detected.

Description

technical field [0001] The invention relates to the field of detection of EGFR mutation gene in cancer patients, in particular to a method for detection of EGFR mutation gene based on multiple fluorescent PCR. Background technique [0002] Human epidermal growth factor (epidermal growth factor receptor, EGFR) is a protein tyrosine kinase receptor, which is widely distributed on the surface of mammalian epithelial cells, fibroblasts, glial cells and other cells, and has tyrosine kinase activity. EGFR is one of the members of HER / ErbB family. After EGFR binds to its ligand, it forms a dimer on the cell surface, activates receptor autophosphorylation through the activity of tyrosine kinase, and regulates transcription factors to activate the transcription of genes through the cascade reaction of adapter proteins and enzymes in the cytoplasm. Instructs cell migration, proliferation, differentiation and apoptosis. When EGFR is mutated, EGFR itself or its ligands are overexpress...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858
CPCC12Q1/6858C12Q2535/137C12Q2537/143C12Q2563/107C12Q2527/125
Inventor 徐高连徐宏古宏晨汪琳琳张玲
Owner SHANGHAI MAG GENE NANOTECH CO LTD
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