Detection method and kit for mutation rates of lung cancer mutation sites

Abstract

The invention discloses a method for detecting mutation rate of lung cancer mutation sites. The method comprises the steps that (1) extraction of cfDNA is performed on a plasma sample of peripheral blood of a lung cancer patient to obtain a cfDNA extract; (2) for a lung cancer-associated EGFR L858R site, a first mutant detection probe (the sequence is SEQ ID NO:5) and a first wild type detection probe (the sequence is SEQ ID NO: 6), a first primer (the sequence is SEQ ID NO: 1) and a second primer (the sequence is SEQ ID NO: 2) are designed, and for a lung cancer-associated EGFR 19 exon deletion site, a second mutant detection probe (the sequence is SEQ ID NO: 7) and a second wild type detection probe (the sequence is SEQ ID NO: 8), a third primer (the sequence is SEQ ID NO: 3) and a fourth primer (the sequence is SEQ ID NO: 4) are designed; (3) the cfDNA extract is used as a template for ddPCR detection to obtain an amplification product containing a sensitive mutation site; (4) dataanalysis is performed on the amplification product to obtain the mutation rate of the EGFR sensitive mutation L858R and 19 Del. Thereby, the mutation rates of the EGFR sensitive mutation L858R and 19Del can be obtained sensitively.

Description

[0001] This application is filed on October 24, 2017 , the application number is 201711003578.2 , the name of the invention is A sort of ddPCR detection method and application of EGFR L858R and 19Del in lung cancer divisional application of the patent application. technical field [0002] The invention relates to the fields of medicine and biotechnology, in particular to a detection method and a kit for the mutation rate of lung cancer mutation sites. Background technique [0003] 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 pat...

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Problems solved by technology

[0010] 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 false positive detection results, especially the cfDNA false positive detection rate in plasma samples is higher, because cfDNA is mainly derived from benign cells of germline origin. The proportion of ctDNA is relatively low; fourth, there are literatures showing that the addition of carrierRNA in the cfDNA extraction process does not increase the total amount of cfDNA extraction, and it is likely to cause large fragments of gDNA to aggregate

Fourth, the steps of liquid biopsy are cumbersome, the cycle is long, and the cost is high, so it is not suitable for the detection of single or two sites

[0011] In summary, circulating tumor DNA-based detection technology has become the focus, but there is still a lack of low-input, high-sensitivity, and high-accuracy mutation DNA detection technology to detect lung cancer EGFR L858R and 19Del

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