DdPCR detection method for lung cancers EGFR L858R and 19Del and application of ddPCR detection method

A detection method, lung cancer technology, applied in the fields of medicine and biology, can solve the problems of low detection sensitivity, increase in the total amount of cfDNA extraction, high cost, etc., and achieve the effect of high sensitivity

Inactive Publication Date: 2018-02-06
深圳海普洛斯医学检验实验室
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AI Technical Summary

Problems solved by technology

[0009] The existing detection methods for lung cancer have the following defects and deficiencies: First, the traditional detection optimization method mainly relies on the experience of technicians to interpret the results, which cannot directly, objectively and accurately reflect the detection results, nor can it perform absolute quantification of DNA; , the existing detection optimization method based on lung cancer EGFR L858R and 19Del usually has high requirements on the total amount and quality of cfDNA in the sample, and is easily affected by a large amount of blood-related DNA and response inhibitors; thirdly, the existing detection method based on lung cancer EGFR L858R and 19Del The detection optimization method of the method is cumbersome, and the detection sensitivity is low, and there will be a certain amount of...

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  • DdPCR detection method for lung cancers EGFR L858R and 19Del and application of ddPCR detection method
  • DdPCR detection method for lung cancers EGFR L858R and 19Del and application of ddPCR detection method
  • DdPCR detection method for lung cancers EGFR L858R and 19Del and application of ddPCR detection method

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Experimental program
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Effect test

Embodiment 1

[0065] Example 1 cfDNA extraction optimization

[0066] The content of ctDNA in peripheral blood is very low, the ratio is about 1 / 1000, so it is very important to ensure the quality of plasma cfDNA extraction.

[0067] Preferably, the detailed steps of the cfDNA extraction scheme are as follows:

[0068] 1. Take 2 mL of peripheral blood and process it within 3 hours after obtaining the blood, including the following steps:

[0069] (1) 1600g, centrifuge peripheral blood for 10min, separate into blood cells and plasma (supernatant), and store blood cells at -80°C;

[0070] (2) The plasma sample was centrifuged for the second time at 16,000 g for 10 min, and the supernatant was transferred to a storage tube and stored at -80°C.

[0071] 2. In this example, nucleic acid extraction or purification (ZD-YL-Midi-40) was used for cfDNA extraction. The optimized extraction steps are as follows:

[0072] (1) Take a clean 10mL centrifuge tube, add 10μL ProteinaseK, then add 1mL plasma ...

Embodiment 2

[0085] Example 2 Detection of EGFR L858R and 19Del loci of ctDNA extracted from peripheral blood of patients with lung cancer

[0086] The droplet digital PCR system (droplet digital PCR) performs droplet treatment on samples before traditional PCR amplification, and divides the reaction system containing nucleic acid molecules into tens of thousands of nanoliter droplets, each of which may or may not Contain nucleic acid target molecules to be detected, or contain one to several nucleic acid target molecules to be detected. After PCR amplification, the fluorescence signal of each droplet is analyzed one by one. The droplet with fluorescent signal is interpreted as 1, and the droplet without fluorescent signal is interpreted as 0. According to the principle of Poisson distribution and the number of positive droplets The initial copy number or concentration of the target molecule can be obtained by ratio. Compared with the traditional PCR technology, the droplet digital PCR me...

Embodiment 3

[0124] Example 3 Analysis of fluorescence signal values ​​at EGFR L858R and EGFR 19Del sites

[0125] 1. Fluorescence signal analysis of EGFR L858R locus

[0126] (1) Open the QuantaSoft software, import the sample detection data, and select the Analyze module to start analyzing the data.

[0127] (2) First of all, it is necessary to confirm that the number of droplets generated by all sample types must be above 10,000, and only the CV value of the data detected within this range can be controlled within 1.6%. Then select 1D Amplitude to determine the fluorescence threshold of the mutant probe and the wild-type probe, and use the blank control group and the wild-type control group to delineate the threshold of the mutant probe and the wild-type probe. figure 2 , image 3 It shows that the EGFR L858R probe designed in the present invention has good specificity, and can clearly distinguish wild-type micro-droplets from mutant-type micro-droplets.

[0128] (3) The QuantaSoft ...

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Abstract

The invention relates to the technical field of medicines and biology and particularly relates to a ddPCR detection method for lung cancers EGFR L858R and 19Del and application of the ddPCR detectionmethod. The detection method comprises the steps of separating hemocyte and plasma of peripheral blood, carrying out cfDNA extraction by virtue of a two-step purification method, respectively designing two probes and one pair of primers aiming at lung cancer-relevant EGFR L858R loci and EGFR 19# exon deletion loci, carrying out ddPCR detection by taking extracted cfDNA as a template, and finally carrying out data analysis on an obtained amplification product, so as to obtain the proportion of EGFR sensitively-mutated L858R and 19Del. The application comprises the application of the ddPCR detection method in the treatment of tumor patients, medication-relevant genetic locus mutation information is provided for the tumor patients in the treatment process of the terminal lung cancer, and important reference frame is provided for the individualized accurate medication guidance, pesticide effect evolution and treatment effect monitoring of the lung cancers.

Description

technical field [0001] The invention relates to the fields of medicine and biotechnology, in particular to a ddPCR detection method and application of lung cancer EGFR L858R and 19Del. Background technique [0002] Lung cancer is the most common primary malignant tumor of the lung. The vast majority of lung cancers originate from the bronchial mucosal epithelium, so it is also called bronchial lung cancer. Over the past 50 years, the morbidity and mortality of lung cancer have risen rapidly in countries all over the world, especially industrially developed countries. It is the malignant tumor with the highest morbidity and mortality in the world. The 5-year survival rate is only 16.8%. The number of patients has reached 1.6 million. Lung cancer is also the leading cause of death of cancer patients, and 1.4 million people die of lung cancer every year. [0003] At present, the traditional lung cancer treatment methods include: surgery, radiotherapy and chemotherapy. Althoug...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858C12Q1/6886
CPCC12Q1/6858C12Q1/6886C12Q2600/106C12Q2600/156C12Q2531/113C12Q2563/107
Inventor 刘园园王一杏张晓妮
Owner 深圳海普洛斯医学检验实验室
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