Development and validation of an ultrasensitive qPCR method to identify and quantify EGFR T790M in cell-free DNA

BACKGROUND: Circulating tumor DNA (ctDNA) is a promising biomarker for cancer prognosis and drug development. A major challenge in the ctDNA determination method is discriminating ctDNA from highly similar but significantly more abundant wild-type DNA sensitively and accurately. METHOD: An ultrasensitive qPCR method termed Triple Enrichment Amplification of Mutation PCR (TEAM-PCR) was developed to detect EGFR T790M mutation. RESULTS: wild-type copies. This method was fully validated following the essential bioanalysis guidance, with the limit of detection (LOD) being five copies/reaction. CONCLUSION: This study established and validated a qPCR-based strategy to detect EGFR T790M mutation with ultra-high sensitivity and reliability.