Probes, primers and kit for detecting T790M mutation of EGFR gene

A P-T790M, kit technology, applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of limited efficacy judgment, inability to perform detection, and high false negative rate. The effect of increasing the detection rate, fast detection speed and simple operation

Inactive Publication Date: 2015-12-02
AMOY DIAGNOSTICS CO LTD
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  • Summary
  • 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 in plasma or blood, the tumor DNA content is often lower than 1%, ARMS-PCR The method is not enough to test the blood, which limits the judgment of clinicians on the efficacy of targeted drugs
In addition, the drug resistance mutations produced in the course of clinical drug use cannot be detected by previous methods due to insufficient sensitivity

Method used

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  • Probes, primers and kit for detecting T790M mutation of EGFR gene
  • Probes, primers and kit for detecting T790M mutation of EGFR gene
  • Probes, primers and kit for detecting T790M mutation of EGFR gene

Examples

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Embodiment 1

[0027] In this embodiment, the system of the present invention is tested with cell lines, the H1975 cell line mutation is positive, and the negative plasma sample is used as a control. The method for detecting EGFR gene T790M mutation by real-time fluorescent PCR of the present invention comprises the steps:

[0028] (1) Test sample processing and DNA extraction:

[0029] The DNA extraction method of the cell line is as follows: add proteinase K and BufferATL, digest and lyse at 56°C for 1 hour, add 200mL BufferAL and mix well, then add 200μL absolute ethanol to mix well, transfer the supernatant carefully to a QIA2mL spin column, 6000×g ( Centrifuge at 8000rpm) for 1min, add 500μL BufferAW1, centrifuge at 6000×g (8000rpm) for 1min, carefully open the lid and add 500μL BufferAW2, centrifuge at 6000×g (8000rpm) for 1min, centrifuge the empty tube at 20000×g (14000rpm) for 3min, add 100μL BufferATE to the center of the membrane, Incubate for 5 minutes and centrifuge at 20,000×g...

Embodiment 2

[0044] A total of 86 cases of EGFR gene T790M mutation 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:

[0045] (1) Sample processing and DNA extraction:

[0046] 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 of pre-cooled isopropanol, upside down and mix well, 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℃ for 10min; cool to room temperature, add 200μL anhydrous Ethanol, blow and mix the precipitate; transfer all the precipitate mixture into the adsorption column, centrifuge at 10,000×...

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Abstract

The invention discloses probes, primers and a kit for detecting a T790M mutation of an EGFR gene. The probes and the primers have the following sequence: SEQ ID NO: 01 to SEQ ID NO. 07. The probes, the primers and the kit have the following benefits: (1) SNP sites on the primers are designed as G/A merged basic groups, so that all efficiencies are compatible, and the amplification efficiency is improved; (2) the sensitivity is high, that is, the detection sensitivity can reach 2 permillage; (3) compared with those adopting a digital PCR method, the operation is simple, the cost is reduced, and the clinical application range is wide; (4) through blood plasma sample detection with a large reaction volume, the DNA loading quantity of blood plasma samples is increased, the detection system is more stable, and the detection rate of the blood plasma samples is improved; (5) the detection speed is high, that is, the detection process can be completed within only 120 minutes, and the time consumed in the detection process is only a half of that consumed according to the digital PCR method; (6) the probes, the primers and the kit, provided by the invention, can be utilized for detecting peripheral blood samples, so that convenient sampling and dynamic detection can be realized.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a probe, a primer and a kit for detecting the mutation of exon 20T790M of epidermal growth factor EGFR gene. Background technique [0002] The human epidermal growth factor receptor family (epidermal growth factor receptor family, EGFR family) belongs to the tyrosine kinase receptor family, also known as the HER family or the erbB family. The EGFR protein tyrosine kinase functional region is encoded by exons 18-24 of the EGFR gene, wherein exons 18-20 encode N-Lobe, and exons 21-24 encode C-Lobe. The EGFR mutations discovered so far are mainly located in exons 19 to 21. The T790M mutation on exon 20 is one of the more recognized drug resistance mechanisms at present. It is usually an alternative point mutation. The C mutation at position 2369 is T . Therefore, in addition to sensitive mutations, the detection of T790M drug-resistant genes is also important in the selection of clini...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q2531/113C12Q2535/137
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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