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

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. An ultrasensitive qPCR method termed Triple Enrichment Amplification of Mutation PCR (TEAM-PCR) was developed to detect EGFR T790M mutation. EGFR T790M was quantified over the assay range of 25–10⁶ copies/reaction in the presence of 10⁶ wild-type copies. This method was fully validated following the essential bioanalysis guidance, with the limit of detection (LOD) being five copies/reaction. This study established and validated a qPCR-based strategy to detect EGFR T790M mutation with ultra-high sensitivity and reliability.