Human EGFR gene mutation site T790M detection reagent and detection method

A technology of T790M and detection reagents, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., which can solve the problems of long cycle, high cost, and difficult clinical promotion, and achieve the effect of trace detection

Pending Publication Date: 2019-10-25
WENZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The sensitivity of Super ARMS PCR is about 0.2-0.8%. ddPCR and NGS can detect mutations as low as 0.1%. High, long cycle, high cost and other disadvantages, it is not easy to promote clinically

Method used

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  • Human EGFR gene mutation site T790M detection reagent and detection method
  • Human EGFR gene mutation site T790M detection reagent and detection method
  • Human EGFR gene mutation site T790M detection reagent and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0107] 1. Preparation of PCR reaction solution I and single base extension solution I

[0108] According to the wild-type sequence of EGFR gene (NC_000007.13) published by NCBI database, and taking the upstream and downstream 75 bp of EGFR p.T790M mutation site as a reference, design specific EGFR amplification primers; mutation site EGFR (c.2369C>T).

[0109] The length of the PCR amplification primer is 28 bases, and a total of 10 bases of ACGTTGGATG are added to the 5' end to increase the molecular weight of the entire PCR primer to distinguish the extension product. The 18 bases are completely complementary to the EGFR DNA in the sample to be tested.

[0110] The extension product sequence is located on the PCR product sequence and is complementary to the sequence upstream of the mutation site.

[0111] Primer sequences for detecting p.T790M mutation of human EGFR gene:

[0112] PCR amplification upstream primer: 5'-ACGTTGGATGATCTGCCTCACCTCCACC-3';

[0113] PCR amplifica...

Embodiment 2

[0129] 1. Detection method of human EGFR gene mutation site T790M

[0130] Schematic diagram of EGFR p.T790M mutation detection principle figure 1 .

[0131] 1) Sample preparation: Paraffin-embedded tissues, fresh tissues, frozen-embedded tissues, blood, plasma and pleural fluid were routinely processed to extract DNA samples.

[0132] 2) PCR amplification of the target fragment: add PCR reaction solution I, PCR reaction solution II, and DNA sample to the reaction tube to prepare a PCR reaction system; set the PCR reaction conditions on the PCR machine to perform PCR. Prepare the PCR reaction system (20μL) according to the dosage of the components in the table below: add DNA samples according to the actual requirements, the volume is 2-10μL, and make up to 20μL with water.

[0133]

[0134]

[0135] PCR reaction conditions: pre-denaturation at 95°C for 15 min; denaturation at 95°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 60 s, and 45 cycles; final ex...

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Abstract

The invention relates to a human EGFR gene mutation site T790Mdetection reagent and detection method. The detection reagent is prepared from a PCR reaction solution I, a PCR reaction solution II, an enzymatic digestion reaction solution,single base extension liquid I, single base extension liquid II and a magnetic particle suspension. The detection method based on the reagent comprises the steps that the PCR reaction solution I and the PCR reaction solution II amplifyan EGFR genein a blood sample, the enzymatic digestion reaction solutionis added for digestion, and the single base extension liquid I, the single base extension solution II and a PCR-SAP product are added into the digested product for single base extension, and product magnetic beads are gathered and then subjected to chip analysis. According to the detection reagent and method used for detecting human EGFR gene p.T790 mutation, as little as 10 mutant DNAs can be detected from 10<4> DNA molecules, the sensitivity reaches0.1%, and T790M micro-detection is achieved.

Description

technical field [0001] The invention relates to the technical field of detection of human EGFR gene mutation site p.T790M (c.2369C>T), in particular to PCR technology and single-base extension reaction, combined with magnetic particle technology, to realize the trace detection of p.T790M. Background technique [0002] EGFR is a glycoprotein receptor on the surface of the cell membrane, has tyrosine kinase (Tyrosine Kinase, TK) activity, and is the expression product of the proto-oncogene c-erbB-1 (HER-1). The main signal transduction pathways of EGFR are: PI3K-PDK pathway, RAS-RAF-MEK-ERK-MAPK pathway, PLC-γ pathway, JAK-STAT pathway. Through these pathways, extracellular signals are converted into intracellular signals, thereby effectively responding to external signal stimuli, regulating cell growth, proliferation, differentiation, and inhibiting cell apoptosis. [0003] In 2003, the U.S. Food and Drug Administration (FDA) approved AstraZeneca's EGFR small-molecule tar...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886C12Q2600/156
Inventor 金胜男丁春明栾菊
Owner WENZHOU MEDICAL UNIV
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