title: Determining lower limits of detection of digital PCR assays for cancer-related gene mutations.
abstract: Digital PCR offers very high sensitivity compared to many other technologies for processing molecular detection assays. Herein, a process is outlined for determining the lower limit of detection (LoD) of two droplet-based digital PCR assays for point mutations of the epidermal growth factor receptor (EGFR) gene. Hydrolysis probe mutation-detection assays forEGFRp.L858R and p.T790M mutations were characterized in detail. Furthermore, sixteen additional cancer-related mutation assays were explored by the same approach. For theEGFRL8585R assay, the assay sensitivity is extremely good, and thus, the LoD is limited by the amount of amplifiable DNA that is analyzed. With 95% confidence limits, the LoD is one mutant in 180,000 wild-type molecules for the evaluation of 3.3 μg of genomic DNA, and detection of one mutant molecule in over 4 million wild-type molecules was achieved when 70 million copies of DNA were processed. The measured false-positive rate for theEGFRL8585R assay is one in 14 million, which indicates the theoretical LoD if an unlimited amount of DNA is evaluated. For theEFGRT790M assay, the LoD is one mutant in 13,000 for analysis of a 3.3 μg sample of genomic DNA, and the dPCR assay limit sensitivity approaches one mutant in 22,000 wild-type molecules.
authors.affiliation: RainDance Technologies, 749 Middlesex Turnpike, Billerica, MA 01821, USA.
authors.forename: Coren A
authors.initials: CA
authors.lastname: Milbury
authors.affiliation: RainDance Technologies, 749 Middlesex Turnpike, Billerica, MA 01821, USA.
authors.forename: Qun
authors.initials: Q
authors.lastname: Zhong
authors.affiliation: RainDance Technologies, 749 Middlesex Turnpike, Billerica, MA 01821, USA.
authors.forename: Jesse
authors.initials: J
authors.lastname: Lin
authors.affiliation: RainDance Technologies, 749 Middlesex Turnpike, Billerica, MA 01821, USA.
authors.forename: Miguel
authors.initials: M
authors.lastname: Williams
authors.affiliation: RainDance Technologies, 749 Middlesex Turnpike, Billerica, MA 01821, USA.
authors.forename: Jeff
authors.initials: J
authors.lastname: Olson
authors.affiliation: RainDance Technologies, 749 Middlesex Turnpike, Billerica, MA 01821, USA.
authors.forename: Darren R
authors.initials: DR
authors.lastname: Link
authors.affiliation: RainDance Technologies, 749 Middlesex Turnpike, Billerica, MA 01821, USA.
authors.forename: Brian
authors.initials: B
authors.lastname: Hutchison
keywords: Assay sensitivity; Digital PCR; EGFR L858R; EGFR T790M; EGFR, epidermal growth factor receptor; Limit of detection; LoB, limit of blank; LoD, limit of detection; Mutation detection; N, total number of droplet events counted; NMut, number of droplets with only mutated DNA; NWT, number of droplets with only wild-type DNA; PCR, Polymerase Chain Reaction; R, ratio of mutant to wild-type molecules; RFP, average false positive rate (ΛFP/#WT); p, fraction of PCR-positive droplets; ΛFP, average number of false-positive events; λ, average number of targets “loaded” per droplet