The ddPCR assays were performed using the PrimePCR™ ddPCR™ Mutation Detection Assay kit, and the PrimePCR™ ddPCR™ EGFR Exon 19 Deletions Screening Kit (Bio-Rad Laboratories, Hercules, CA, USA) [11 (link)]. Tumor-derived nucleic acids (cfDNA and exoTNA) were extracted from 1 to 2 mL of plasma or pleural fluid. The ddPCR assay for detecting EGFR mutations was validated using the Multiplex I cfDNA Reference Standards (Horizon Discovery, Cambridge, UK) and healthy control samples from a previous study [11 (link)]. Briefly, the amplifications were performed in a reaction volume of 20 μL on a QX100 Droplet Digital PCR System (Bio-Rad). The 20 μL of the PCR mixture was composed of 10 μL Bio-Rad Super mix TaqMan, 1–2 μL of each amplification primer/probe mix, and 8–9 μL of nucleic acids (NAs). Thermal cycling comprised an initial denaturing and polymerase hot-start activating step of 10 min at 95 °C, followed by 40 cycles of 95 °C for 30 s and 55 °C for 60 s. The results were analyzed with QuantaSoft v.1.7.2 software (Bio-Rad) and reported as copies per milliliter of plasma. The ddPCR assay was validated for detecting EGFR mutations and determining the limit of detection (LOD) in a previous study [11 (link)]. The assays were considered “positive” if the measured event rate was ≥ 2 events/assay and “negative” if the event rate within a gated region was < 2 events/assay.
Free full text: Click here
