WO2023109632A1 - Method for detecting egfr gene mutation in circulating tumor cells by using digital pcr and application of method - Google Patents

Abstract

The present invention relates to the technical field of biomedicine. Provided are a method for detecting EGFR gene mutation in circulating tumor cells by using digital polymerase chain reaction (PCR) and an application of the method. The method of the present invention comprises the following steps: (1) enriching circulating tumor cells; (2) extracting the circulating tumor cells to obtain a DNA sample; (3) preparing a PCR system; (4) using a liquid drop generation instrument to prepare the PCR system into liquid drops having uniform sizes; (5) performing PCR amplification on the liquid drops; and (6) performing fluorescence signal detection on the amplified liquid drops. According to the detection method provided by the present invention, the expression of an EGFR in a patient with advanced or recurrent non-small cell lung cancer can be detected without obtaining, by means of a needle biopsy, a tissue sample.

Description

A method for detecting EGFR gene mutation in circulating tumor cells by digital PCR and its application technical field

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

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.

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, recurrence and prognosis of cancer patients. Using them to detect EGFR mutations in patients will help to monitor tumor metastasis and recurrence, guide targeted drugs, and evaluate drug efficacy and prognosis.

Existing technologies for detecting EGFR mutations in lung cancer patients mainly use patient tissue samples, and use dye method or probe method for detection by fluorescent quantitative PCR technology. However, the detection of EGFR mutations in tissue samples by dye-based or probe-based fluorescent quantitative PCR technology is complicated, takes a long time and has low sensitivity, making it difficult to perform multiple or real-time detections.

technical problem

Existing technologies for detecting EGFR mutations in lung cancer patients mainly use patient tissue samples, and use dye method or probe method for detection by fluorescent quantitative PCR technology. However, the detection of EGFR mutations in tissue samples by dye-based or probe-based fluorescent quantitative PCR technology is complicated, takes a long time and has low sensitivity, making it difficult to perform multiple or real-time detections.

technical solution

Aiming at the problems existing in the above-mentioned prior art, the object of the present invention is to design and provide a method for detecting EGFR gene mutation in circulating tumor cells by digital PCR and its application. Based on the droplet digital PCR technology, the present invention can realize a detection process with lower cost, higher precision, higher sensitivity, lower sample input and absolute quantification, and achieves the purpose of dynamic monitoring.

In order to achieve the above object, the present invention adopts the following technical solutions:

A method for detecting EGFR gene mutations in circulating tumor cells by digital PCR, characterized in that DNA samples are extracted from circulating tumor cells, a PCR reaction system is configured to generate droplets, PCR amplification is performed, and EGFR gene mutations are detected with fluorescent signals.

Described method is characterized in that comprising the following steps:

(1) enrich circulating tumor cells;

(2) Extract the circulating tumor cells enriched in the above step (1) to obtain DNA samples;

(3) Configure the PCR reaction system;

(4) Using a droplet generation instrument to prepare the PCR reaction system obtained in the above step (3) into droplets of uniform size;

(5) performing PCR amplification on the droplets generated in the above step (4);

(6) Fluorescent signal detection is performed on the droplets amplified in the above step (5).

The method for detecting EGFR gene mutation in circulating tumor cells by digital PCR is characterized in that in the step (1), the method of separating peripheral blood samples by a deterministic lateral displacement chip is used to enrich circulating tumor cells.

The method for detecting EGFR gene mutations in circulating tumor cells by digital PCR is characterized in that the PCR reaction system in step (3) includes: 1 μL of target primers/probes, 1 μL of control primers/probes, PCR 4 μL of the premix, 10 μL of the DNA sample obtained in the step (2), supplemented with water, and 20 μL of the total system.

The method for detecting EGFR gene mutation in circulating tumor cells by digital PCR is characterized in that the primers include wild type and mutant type, and the fluorescent probe includes wild type and mutant type.

The method for detecting EGFR gene mutation in circulating tumor cells by digital PCR is characterized in that the size of the droplet in the step (4) is 80-100 μm.

The method for detecting EGFR gene mutation in circulating tumor cells by digital PCR is characterized in that the amplification conditions in step (5) are: 95°C for 10 min; 94°C for 30 s, 55°C for 60 s, 40°C cycle; store at 20°C.

The method for detecting EGFR gene mutation in circulating tumor cells by digital PCR is characterized in that the fluorescent signal detection in the step (6) is performed on a tiled chip.

The method for detecting EGFR gene mutation in circulating tumor cells by digital PCR is characterized in that the height of the tiling chip is 100-110 μm.

The application of the method in the detection of EGFR gene mutation in circulating tumor cells.

Beneficial effect

Compared with the prior art, the present invention has the following beneficial effects:

(1) The detection method provided by the present invention can detect the expression of EGFR in patients with advanced or recurrent non-small cell lung cancer without obtaining tissue samples by needle biopsy. Feasibility in clinical application.

(2) The method of the present invention has the characteristics of high sensitivity, high accuracy, low cost, etc., and is easy to operate.

Description of drawings

Figure 1 is the droplet PCR detection results (10X) of the EGFR gene T790M mutation.

Embodiments of the present invention

The present invention will be further described with reference to the accompanying drawings and examples below.

Example 1:

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

(1) Enrich circulating tumor cells by separating peripheral blood samples using a deterministic lateral displacement chip.

(2) Extract the circulating tumor cells enriched in the above step (1) to obtain DNA samples.

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

(4) Prepare the obtained PCR reaction system into uniformly sized droplets with a droplet generation instrument, and the size of the droplets is 80-100 μm.

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

(6) Fluorescent signal detection is performed on the droplets amplified in the above step (5). Fluorescent signal detection is carried out on a tiled chip, and the height of the tiled chip is 100-110 μm. Successful detection of the EGFR gene T790M mutation signal, as shown in Figure 1, proves the feasibility of the present invention.

Claims (10)

A method for detecting EGFR gene mutations in circulating tumor cells by digital PCR, characterized in that DNA samples are extracted from circulating tumor cells, a PCR reaction system is configured to generate droplets, PCR amplification is performed, and EGFR gene mutations are detected with fluorescent signals. The method of claim 1, comprising the steps of: (1) enrich circulating tumor cells; (2) Extract the circulating tumor cells enriched in the above step (1) to obtain DNA samples; (3) Configure the PCR reaction system; (4) Using a droplet generation instrument to prepare the PCR reaction system obtained in the above step (3) into droplets of uniform size; (5) performing PCR amplification on the droplets generated in the above step (4); (6) Fluorescent signal detection is performed on the droplets amplified in the above step (5). A method for detecting EGFR gene mutations in circulating tumor cells by digital PCR as claimed in claim 2, characterized in that in said step (1), the method of separating peripheral blood samples by a deterministic lateral displacement chip is used to enrich circulating tumor cells . A method for detecting EGFR gene mutations in circulating tumor cells by digital PCR according to claim 2, characterized in that the PCR reaction system in the step (3) includes: 1 μL of target primers/probes, control primers/probes 1 μL, 4 μL of PCR master mix, 10 μL of the DNA sample obtained in step (2), supplemented with water, and 20 μL of the total system. A method for detecting EGFR gene mutation in circulating tumor cells by digital PCR according to claim 4, characterized in that said primers include wild type and mutant type, and said fluorescent probe includes wild type and mutant type. A method for detecting EGFR gene mutations in circulating tumor cells by digital PCR according to claim 2, characterized in that the size of the droplets in the step (4) is 80-100 μm. A method for detecting EGFR gene mutations in circulating tumor cells by digital PCR according to claim 2, characterized in that the amplification conditions in the step (5) are: 95°C for 10 min; 94°C for 30 s, 55°C 60 s, 40 cycles; store at 20°C. A method for detecting EGFR gene mutations in circulating tumor cells by digital PCR according to claim 2, characterized in that the fluorescent signal detection in the step (6) is performed on a tiled chip. A method for detecting EGFR gene mutation in circulating tumor cells by digital PCR according to claim 8, characterized in that the height of the tiled chip is 100-110 μm. Application of the method according to any one of claims 1-9 to the detection of EGFR gene mutation in circulating tumor cells.