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Method for detecting EGFR (epidermal growth factor receptor) gene mutation of circulating tumor cells by digital PCR (polymerase chain reaction) and application of method

A tumor cell and digital technology, applied in the field of biomedicine, can solve the problems of difficult multiple or real-time detection, long detection time, low sensitivity, etc., and achieve the effect of high accuracy, convenient operation, and high sensitivity

Pending Publication Date: 2022-04-26
SHENZHEN INST OF ADVANCED TECH
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Problems solved by technology

However, the detection of EGFR mutations in tissue samples by dye method or probe method by fluorescent quantitative PCR technology is complicated, the detection time is long and the sensitivity is not high, and it is difficult to achieve multiple or real-time detection.

Method used

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  • Method for detecting EGFR (epidermal growth factor receptor) gene mutation of circulating tumor cells by digital PCR (polymerase chain reaction) and application of method
  • Method for detecting EGFR (epidermal growth factor receptor) gene mutation of circulating tumor cells by digital PCR (polymerase chain reaction) and application of method

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Embodiment 1

[0029] A method for detecting EGFR gene mutation in circulating tumor cells by digital PCR, comprising the following steps:

[0030] (1) The method of deterministic lateral displacement chip separation of peripheral blood samples was used to enrich circulating tumor cells.

[0031] (2) Extracting the circulating tumor cells enriched in the above step (1) to obtain a DNA sample.

[0032] (3) Configure the PCR reaction system. The configuration of the PCR reaction system is shown in Table 1 below.

[0033] Table 1 PCR reaction system configuration

[0034]

[0035] (4) Prepare the obtained PCR reaction system into droplets with a uniform size by using a droplet generation instrument, and the size of the droplets is 80-100 μm.

[0036] (5) Perform PCR amplification on the droplets generated in the above step (4). Amplification conditions were: 95°C for 10 min; 94°C for 30 s, 55°C for 60 s, 40 cycles; 20°C storage, as shown in Table 2 below.

[0037] Table 2 Amplification ...

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Abstract

The invention relates to a method for detecting EGFR (epidermal growth factor receptor) gene mutation of circulating tumor cells by digital PCR (polymerase chain reaction) and application thereof, belonging to the technical field of biomedicine. The method comprises the following steps: (1) enriching circulating tumor cells; (2) extracting the circulating tumor cells to obtain a DNA sample; (3) preparing a PCR reaction system; (4) preparing the PCR reaction system into liquid drops with uniform sizes by adopting a liquid drop generation instrument; (5) carrying out PCR (Polymerase Chain Reaction) amplification on the liquid drops; and (6) carrying out fluorescence signal detection on the amplified liquid drops. According to the detection method provided by the invention, the EGFR expression condition of a patient with advanced or recurrent non-small cell lung cancer can be detected without acquiring a tissue specimen through needle biopsy, the technology is non-invasive and can be used for multiple times of real-time detection, and the feasibility of circulating tumor cells in clinical application is improved. The method has the characteristics of high sensitivity, high accuracy, low cost and the like, and is convenient to operate.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a method for detecting EGFR gene mutation of circulating tumor cells by digital PCR and an application thereof. Background technique [0002] Epidermal growth factor (EGFR) is a transmembrane protein, which can provide the basis for the selection of targeted drugs and therapeutic targets in patients with non-small cell lung cancer after surgery. With the discovery of EGFR and the development and clinical application of molecular targeted therapy drugs, the 5-year survival rate of lung cancer patients has improved to a certain extent. [0003] Circulating tumor cells refer to tumor cells that are released from solid tumors or metastases into the peripheral blood circulation spontaneously or due to diagnostic and therapeutic operations. A large number of experiments have proved that circulating tumor cells in peripheral blood are closely related to the metastasis, ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/686
CPCC12Q1/6886C12Q1/686C12Q2600/156C12Q2563/107C12Q2563/159
Inventor 陈艳黄玉清
Owner SHENZHEN INST OF ADVANCED TECH
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