Probes, primers, detection system and kit for detecting mutations of EGFR gene

A P-EGFR, kit technology, applied in the detection system and kits, primers, and probes for epidermal growth factor gene mutation detection, can solve the problems of low sensitivity, complicated operation, blood detection, etc., and achieve high sensitivity and specificity. Good performance and high accuracy

Active Publication Date: 2016-02-24
AMOY DIAGNOSTICS CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The sequencing method is about 20% less sensitive, and the operation is complicated, the detection time is longer, and the false negative rate is higher
The ARMS-PCR method can achieve a sensitivity of 1%, which can meet the detection requirements for tumor tissue samples, but for blood samples that are convenient to sample, such as circulating tumor cells

Method used

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  • Probes, primers, detection system and kit for detecting mutations of EGFR gene
  • Probes, primers, detection system and kit for detecting mutations of EGFR gene
  • Probes, primers, detection system and kit for detecting mutations of EGFR gene

Examples

Experimental program
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Effect test

Embodiment 1

[0051] In this example, the plasmid constructed by genetic engineering was used as the positive plasmid, and the negative plasma sample was used as the control. Utilize real-time fluorescent PCR of the present invention to detect the method for EGFR25 kinds of gene mutations to comprise the steps:

[0052] (1) Test sample processing and DNA extraction:

[0053] Plasmid processing and extraction: each plasmid was extracted using a plasmid extraction kit, and the specific extraction steps were operated according to the kit instructions. The extracted DNA was dissolved in Tris-HCl (10mmol / L, pH8.0), and the quality of the extraction was detected by a UV spectrophotometer to determine its concentration. Then, the DNA concentration was adjusted to different copy numbers with negative plasma DNA, and used as a PCR template. 15 μL for PCR reaction amplification.

[0054] The plasma DNA extraction method is as follows: pipette 4mL sample into a 10mL round bottom centrifuge tube, add...

Embodiment 2

[0079] A total of 97 cases of EGFR gene mutations in clinical plasma samples were detected by using the present invention, and compared with digital PCR detection. Utilize specific primer of the present invention and probe fluorescent PCR system to detect steps as follows:

[0080] (1) Sample processing and DNA extraction:

[0081] Pipette 4mL sample into a 10mL round-bottom centrifuge tube, add 1.8mL BufferCDL, then add 50μL ProfeinaseKSolution, mix well for 10s; put it in a water bath, digest at 63°C for 15min, quickly cool to room temperature, add 400μL DNATracer, mix well, then add 3.3mL pre-cooled isopropanol, mix upside down, centrifuge at 10000×g for 5min; add 470μL BufferCDB and 10μL ProteinaseKSolution to the precipitate, shake well, put it in a water bath, digest at 63°C for 10min; cool to room temperature, add 200μL absolute ethanol , pipette and mix the precipitate; transfer all the precipitate mixture into the adsorption column, centrifuge at 10000×g for 30s, pou...

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Abstract

The invention discloses probes, primers, a detection system and a kit for detecting mutations of an EGFR gene, and the probes and the primers have the sequences of SEQ ID NO.1 to SEQ ID NO.24. The invention is characterized in that (1), the amplification efficiency is greatly improved to a largest extent; (2) the sensitivity is high, and the detection sensitivity can reach 0.2%; (3) compared with a digital PCR method, operations are simple, the cost is saved, and the clinical application scope is wide; (4) plasma samples with large reaction volume are detected, the DNA sampling quantity of the plasma samples becomes larger, the system is more stable, and the detection rate of the plasma samples is increased; (5) 25 mutations of the EGFR gene can be detected simultaneously in three reaction tubes, and results are intuitive and clear; (6) the detection speed is fast, and consumed time is only 1/2 that of the digital PCR; and (7) a detection method can detect peripheral blood samples, has convenient sampling, and can dynamically monitor curative effect of EGFR-TKI drugs on patients.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a probe, a primer, a detection system and a kit for detection of epidermal growth factor (EGFR) gene mutation. Background technique [0002] The epidermal growth factor receptor gene is located on the short arm of human chromosome 7 and consists of 188,307 bases, including 28 exons. Its tyrosine kinase domain is encoded by exons 18-24. Epidermal growth factor receptor (EGFR) is the expression product of proto-oncogene C-erbB-1 (HER-1), located on the cell membrane. EGFR mainly transmits signals to the nucleus through two pathways, one is Ras→Raf→MAPK pathway; the other is PI3K→PKC→IKK pathway. When the signal is transmitted to the nucleus, it will cause an increase in the transcription level of genes in the nucleus, resulting in cell proliferation and transformation. Abnormality of EGFR signaling is the cause of many tumors. Epidermal growth factor receptor tyrosine kinase inhibi...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6886C12Q2600/106C12Q2600/156
Inventor 江风阁林清华陈婷宋庆涛阮力朱冠山郑立谋
Owner AMOY DIAGNOSTICS CO LTD
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