Circulating tumor DNA EGFR detecting technology and reagent kit thereof

Abstract

The invention discloses a circulating tumor DNA EGFR (Epidermal Growth Factor Receptor) detecting technology and a reagent kit thereof. The circulating tumor DNA EGFR detecting technology comprises the following steps: step 1, designing a synthetic primer, a closed probe and a detecting probe for a mutation site; step 2, processing a sample and extracting DNA; step 3, applying the circulating tumor DNA EGFR detecting technology to the reagent kit, and establishing a PCR amplified reaction system. According to the circulating tumor DNA EGFR detecting technology disclosed by the invention, the specific MGB Blocker probe, the EGFR specific probe and the primer are adopted to detect the EGFR mutation in the circulating tumor DNA; the MGB Blocker probe is utilized to retard nonspecific amplification of the EGFR wild type genome and improves the sensitivity; the circulating tumor DNA EGFR detecting technology can be used for detecting the EGFR mutation in the circulating tumor DNA, is simple to operate, cheap in detection, capable of being popularized at a large scale and high in detection speed; the detecting process can be completed for about 2 hours.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a circulating tumor DNA EGFR detection technology and a kit thereof. Background technique [0002] Epidermal growth factor receptor (EGFR) is a member of the human epidermal growth factor receptor (HER) family, belongs to the receptor tyrosine kinase family, and is the expression product of the proto-oncogene c-erbB1. EGFR is mainly located in the cell membrane, and after being activated by ligands such as EGF, dimerization occurs and triggers phosphorylation of the intracellular segment, generating tyrosine kinase activity and further activating downstream signaling pathways (Pao W et al.Proc Natl Acad Sci US A.2004 ; 101:13306-11), regulating cell growth, proliferation and other physiological processes. EGFR downstream signaling pathways mainly include: Ras / Raf / MEK / ERK / MAPK pathway, PI3K / PDK1 / Akt pathway, PLC-γ pathway, JAK / STAT pathway, etc. (Sun J M et al.Lung Cancer.2010; 68:42...

AI Technical Summary

Problems solved by technology

[0007]At present, EGFR mutation detection is based on tumor tissue samples, so there are two problems. First, most tumor tissues come from biopsy techniques, namely puncture and biopsy forceps biopsy. This will bring great pain to the patient and is an invasive test

Second, for some advanced patients, puncture or surgery is not possible, and it is difficult to obtain patient samples, so it is difficult to target patients with drugs based on the test results

However, due to its complicated operation and expensive equipment, it is not suitable for large-scale clinical promotion.

Digital PCR is another high-sensitivity detection technology that has emerged in recent years. This technology can reach the highest sensitivity of 0.01%, but this technology is very prone to false positive results (Pinheiro LB et al.2012Anal Chem; 84( 2):1003–1011)

Similarly, high equipment and reagent prices and extremely high experimental operation requirements also limit its large-scale promotion

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