Primers, probes, kit and method for detecting human EGFR (epidermal growth factor receptor) gene mutations

A kit and human technology, applied in biochemical equipment and methods, microbiological measurement/testing, DNA/RNA fragments, etc., can solve problems such as difficulty in popularization, poor effect, and low sensitivity

Active Publication Date: 2012-10-24
武汉海吉力生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The direct sequencing method is time-consuming and has low sensitivity. It is usually only used in scientific research and is difficult to use in clinical detection.
The traditional fluorescent quantitative PCR method is difficult to specifically amplify the mutant gene in a large number of wild-type genes, and it is easy to produce false positive detection results
The high-resolution melting curve method uses special instruments, which are difficult to popularize in hospitals, and this method is less effective in detecting deletion mutations

Method used

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  • Primers, probes, kit and method for detecting human EGFR (epidermal growth factor receptor) gene mutations
  • Primers, probes, kit and method for detecting human EGFR (epidermal growth factor receptor) gene mutations

Examples

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Effect test

Embodiment 1

[0075] Embodiment 1: Extraction of clinical sample DNA

[0076] In this example, DNA was extracted from paraffin-embedded tissue sections of the lungs of patients with non-small cell lung cancer, and was quantified as a template for PCR detection. The QIAamp paraffin-embedded tissue extraction kit from Qiagen was used, as detailed below.

[0077] 1. Preparation before experiment

[0078] (1) Adjust the water bath to 56°C.

[0079] (2) Place Buffer ATL and Buffer AL in the QIAamp kit at 56°C until the precipitate dissolves.

[0080] 2. Sample processing

[0081] (1) Cut off the excess paraffin on the paraffin section, cut the section into a suitable size, and install it on a paraffin microtome.

[0082] (2) Adjust the caliper to 10 μm, and then cut the wax block until a uniform tissue can be clearly seen on the cut section.

[0083] (3) Get rid of the first 5 pieces, start from the 6th piece, put them into 1.5ml EP tubes, 4 pieces in each tube, and then number them.

[...

Embodiment 2

[0102] Example 2: Real-time fluorescent PCR method to amplify clinical sample DNA

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Abstract

The invention provides primers, probes, kit and method for detecting human EGFR (epidermal growth factor receptor) gene mutations. The method comprises the following steps of: (1) providing the primers and the probes; (2) processing detected samples and extracting DNA (deoxyribonucleic acid); (3) carrying out fluorescent quantitation on components of a PCR (polymerase chain reaction) system; (4) amplifying target sequences of an EGFR gene mutation to be detected; and (5) distinguishing wild type genes and mutant type genes by utilizing ARMS (amplification refractory mutation system) primers and judging results via the fluorescence intensity of an FAM and a JOE. The primers, probes and method provided by the invention have the beneficial effects that 29 kinds of mutations on the EGFR genes can be simultaneously detected, so that the sensitivity is high, the specificity is strong and the detection speed is high; and the whole detection process only takes 60 minutes.

Description

technical field [0001] The invention belongs to the field of gene mutation detection technology, in particular to primers, probes, kits and detection methods for detecting 29 mutations of human EGFR genes. Background technique [0002] Human epidermal growth factor receptor (EGFR) is a protein tyrosine kinase receptor widely distributed on the surface of mammalian epithelial cells, fibroblasts, glial cells, keratinocytes and other cells, with tyrosine Acid kinase activity. It is a member of the HER / ErbB family, so it is also known as HER1 or ErbB1. After EGFR binds to its ligand, it forms a dimer on the cell surface, activates receptor autophosphorylation through the activity of tyrosine kinase, sends a signal in the cell, and regulates transcription factors through the cascade reaction of adapter proteins and enzymes in the cytoplasm Activates the transcription of genes that direct cell migration, adhesion, proliferation, differentiation and apoptosis. When EGFR is mutat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 赵珊珊喻德华赵平锋
Owner 武汉海吉力生物科技有限公司
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