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Kit for detecting 22 mutations of EGFR genes by utilizing digital PCR technology

An EGFRT790M and kit technology, applied in the fields of biotechnology and medicine, can solve the problems of inability to absolute quantification, poor detection accuracy, insufficient sensitivity, etc., to shorten the experimental operation time and result interpretation time, low cost, and save clinical sample DNA. Effect

Pending Publication Date: 2018-11-20
BEIJING ACCB BIOTECH
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  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

[0010] The purpose of the present invention is to provide a detection primer, probe and kit for detecting 22 mutations of the EGFR gene using digital PCR technology in order to solve the defects of poor detection accuracy, insufficient sensitivity, and inability to be absolutely quantified in the prior art. Detection of EGFR gene L858R point mutation, T790M point mutation, and 20 kinds of 19del deletion mutations in samples (tissue DNA, plasma and urine free DNA), providing a basis for the selection of TKI drugs targeting EGFR

Method used

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  • Kit for detecting 22 mutations of EGFR genes by utilizing digital PCR technology
  • Kit for detecting 22 mutations of EGFR genes by utilizing digital PCR technology
  • Kit for detecting 22 mutations of EGFR genes by utilizing digital PCR technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0112] Embodiment 1 detects the analytical sensitivity (minimum detection limit) of 22 kinds of mutations of EGFR gene 19, 20, 21 exons

[0113] (1) Preparation of detection limit reference product

[0114] a) For the 22 mutant types of exons 19, 20, and 21 of the EGFR gene listed in the table below, through the gene cloning techniques routinely used in the art, design and construct corresponding mutant plasmids according to the sequence of the mutant gene, by The Sanger sequencing method verified that the sequence of the constructed plasmid containing the EGFR mutant gene sequence (hereinafter referred to as "mutant plasmid") met the design requirements.

[0115] b) Select the human lung cancer cell line A549 (where the EGFR gene is wild-type) for in vitro culture, collect the cells and use a cell genome DNA extraction kit to extract the DNA of the A549 cell line, and use a micro-spectrophotometer to measure the concentration of the DNA, according to Determination of concent...

Embodiment 2

[0126] The analytical specificity of embodiment 2 detection reagents

[0127] (1) Preparation of wild type reference product

[0128] a) Select the human lung cancer cell line A549 (where the EGFR gene is wild-type) for in vitro culture, collect the cells and use a cell genome DNA extraction kit to extract the DNA of the A549 cell line, and use a micro-spectrophotometer to measure the concentration of the DNA, according to Determination of concentration DNA was diluted with TE (Tris-HCl EDTA pH 8.0) to 2ng / μl and 5ng / μl respectively.

[0129] b) Genomic DNA can be fragmented into fragmented DNA with a length of 150±50bp using an ultrasonic instrument (Covaris M220) and the parameter configuration in the instruction manual, which is almost consistent with the length distribution of plasma free DNA and can be used as a cfDNA reference product .

[0130] (2) Determination of analytical specificity

[0131] For 2ng / μl, 5ng / μl genomic DNA reference substance or cfDNA reference sub...

Embodiment 3

[0136] Example 3 The changes in the detection primers and probe sequences affect the analytical sensitivity and / or analytical specificity, so the primers and probe sequences involved in the present invention are all optimal combinations, which can ensure that the detection of EGFR genes 19, 20, 21 It has high sensitivity and high specificity when there are 22 mutant types.

[0137] Example 4 Detection of EGFR genes L858R, T790M and 19del in cancer tissue and plasma of 58 patients with advanced non-small cell lung cancer

[0138] (1) Collect paraffin-embedded tissue samples and whole blood samples from 58 patients with advanced non-small cell lung cancer. Among them, the collected whole blood is contained in a special anticoagulant blood collection tube (produced by Streck Company) that can protect the stability of free DNA, and can be stored stably for 7 days at 4-30°C.

[0139] (2) Take 4 paraffin sections (10 μm thick) from each sample, use the paraffin-embedded tissue DNA ...

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Abstract

The invention discloses a kit for detecting 22 mutations of EGFR genes by utilizing a digital PCR technology. The kit comprises at least one of a reagent EGFR 19 del, a reagent EGFR T790M and a reagent EGFR L858R, as well as positive quality control and negative quality control. The kit disclosed by the invention is capable of quantitatively determining L858R point mutation, T790M point mutation and 20 deletion mutations of 19 del of the EGFR genes in samples comprising tissue DNA and free DNA of plasma and urine. The primer, probe and kit disclosed by the invention are high in detection accuracy, high in sensitivity and convenient to operate, and reference is made to TKI type drug choice of the targeted EGFR.

Description

technical field [0001] The invention relates to a kit, in particular to a kit for detecting 22 kinds of mutations of EGFR gene by digital PCR technology, which belongs to the fields of biotechnology and medicine. Background technique [0002] The human epidermal growth factor receptor (Epidermal growth factor receptor, EGFR) gene is located on the short arm of human chromosome 7 (7p12), is about 118kb in length, and consists of 28 exons. The transcribed mRNA is about 5.6kb in length and encodes a transmembrane glycoprotein with a molecular weight of 170kD. The intracellular region has tyrosine kinase (tyrosine kinase, TK) activity and is responsible for transmitting extracellular signals into the cell. Abnormal EGFR activation can promote tumor cell proliferation, migration, differentiation, angiogenesis, and can inhibit tumor cell apoptosis. EGFR forms a homodimer after binding to its ligand, and can also form a heterodimer with other TK receptors, leading to the activatio...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6886C12Q2600/156C12Q2563/159C12Q2535/137
Inventor 莫敏俐陈钊李晖刘玉忠李东霞刘阳
Owner BEIJING ACCB BIOTECH
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