Detection of EGFR gene with a droplet digital PCR chip integrating a double-layer glass reservoir

Abstract

The lack of reliable and practical method for detecting rare hot mutation of epidermal growth factor receptor (EGFR) in circulating tumor DNA (ctDNA) for lung cancer has remained a challenge for general clinical application due to excess wild type DNA in clinical samples. In this study, we developed a droplet digital PCR (ddPCR) platform, integrating a PDMS chip and double-layer glass reservoir. The duplex T-junction droplet generators in PDMS chip can produce about one million uniform droplets of 4.187 pL within ∼10 min, which were then stored in the glass reservoir. The double-layer glass reservoir can protect droplets from evaporation and breaking, solving the problem of instability during thermal-cycling. The quantitative capabilities of the ddPCR chip were evaluated by testing EGFR exon gene 21, with a good linear correlation in the wide range of 10¹ to 10⁶ copies/μL (R² = 0.9998). We then demonstrated that the proposed ddPCR device can recognize rare EGFR L858R mutation under a background of 10⁶ copies/μL wild-type DNA at a sensitivity of 0.0001%. Finally, we demonstrated this ddPCR platform could identify low amount of EGFR L858R mutation in ctDNA and CTCs of patients with lung cancer.

Keywords: Circulating tumor DNA (ctDNA); Double-layer glass reservoir; Droplet digital PCR chip; EGFR L858R mutation; Lung cancer.