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PCR kit for detecting EGFR gene T790M mutation and detection method thereof

A kit and gene technology, applied in the field of PCR kits for detection of EGFR gene T790M mutation, can solve the problems of wide application limitations, rare mutations and high price of structural variation, and achieve the effect of improving sensitivity and quantitative accuracy

Inactive Publication Date: 2019-02-26
上海睿璟生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The ARMS method can only detect known mutations, which is convenient, fast, and highly specific, and is widely used in clinics; while NGS is a high-throughput deep sequencing technology group developed in recent years, which can analyze millions to billions of DNA molecules at a time. Perform parallel sequencing, but it is insensitive to rare mutations and structural variations and expensive, and its wide application is still limited

Method used

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  • PCR kit for detecting EGFR gene T790M mutation and detection method thereof
  • PCR kit for detecting EGFR gene T790M mutation and detection method thereof
  • PCR kit for detecting EGFR gene T790M mutation and detection method thereof

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Embodiment 1

[0040] see Figure 1~2 , a PCR kit for detecting the EGFR gene T790M mutation, comprising nucleic acid amplification reagents and reference substances, the nucleic acid amplification reagent comprising a reaction solution containing specific primers for detecting the EGFR gene T790M mutation and Fluorescent probes, the specific primers also include a pair of upstream primers and downstream primers, the fluorescent probes are MGB fluorescent probes, and the MGB fluorescent probes also include wild-type probes and mutant probes; the The sequences and modifications of specific primers and fluorescent probes are as follows:

[0041] Upstream primer 5'-ACGTGTGCCGCCTGCT-3'

[0042] Downstream primer 5'-TTGTGTTCCCGGACAT-3'

[0043] Wild type probe 5'-FAM-TCATCACGCAGCTC-MGB-3'

[0044] Mutant probe 5'-FAM-TCATCATGCAGCTC-MGB-3';

[0045] The content of the specific primer contained in the above reaction solution is 900nmol / L, and the content of the fluorescent probe is 250nmol / L. ...

Embodiment 2

[0052] see Figure 3-5 , the detection method of the PCR kit that detects EGFR gene T790M mutation as described in embodiment 1, comprises steps as follows:

[0053] (1) According to different sample types, select the corresponding commercial DNA extraction kit for DNA extraction as a template for detection;

[0054] (2) Prepare EGFR T790M detection 3D digital PCR reaction system, which includes:

[0055]

[0056] (3) Using a chip loading instrument, take 14.5 μL of the above reaction system and evenly distribute it on the PCR chip;

[0057] (4) Place the chip on the PCR instrument for amplification, the amplification procedure is:

[0058]

[0059] (5) After the amplification is completed, take out the chip; place the chip in the chip reader for scanning;

[0060] (6) After scanning, upload the data to the server, define the threshold and complete the analysis of the results.

[0061] This kit can detect mutations as low as 0.05% in 20ng of total DNA.

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Abstract

The invention relates to the field of gene mutation detection, and discloses a PCR kit for detecting EGFR gene T790M mutation. The PCR kit comprises a nucleic acid amplification reagent and a reference substance, wherein the nucleic acid amplification reagent comprises EGFR T790M reaction solution which comprises a specific primer and a fluorescent probe for detecting the EGFR gene T790M mutation;the reaction solution further comprises a pair of upstream primer and downstream primer and an MGB fluorescent probe; the nucleic acid amplification reagent further comprises 2*QuantStudio 3D DigitalPCR Mix. The invention furthermore discloses a detection method of the PCR kit for detecting EGFR gene T790M mutation. The kit uses digital PCR technology to quantitatively detect T790M mutations intissues or plasma DNA samples of non-small cell lung cancer patients, thereby assisting the formulation of targeted drug treatment protocols for patients.

Description

technical field [0001] The invention relates to the field of gene mutation detection, in particular to a PCR kit for detecting EGFR gene T790M mutation and a detection method thereof. Background technique [0002] Lung cancer has always been the leading cause of cancer-related deaths worldwide. According to data released by the American Cancer Society, there were 224,210 new cases of lung cancer in the United States in 2014, ranking second among cancers, of which non-small cell lung cancer accounted for 80%. Despite some advances in chemotherapy, the median survival time of the most effective platinum-based chemotherapy regimens does not exceed 10 months. In recent years, the wide application of EGFR tyrosine kinase inhibitors (EGFR-TKI) has greatly improved the treatment of non-small cell lung cancer. At present, the first-generation EGFR tyrosine kinase inhibitors gefitinib and erlotinib are widely used in the treatment of NSCLC. They reversibly bind to the ATP-binding ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886
CPCC12Q1/6886C12Q2600/156
Inventor 包文静金安娜龚伟高伙妮
Owner 上海睿璟生物科技有限公司
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