dPCR assay validation and optimization for concentration levels and thresholds
Serially diluted plasmids for NAP successfully amplified at all concentrations using the FAM labeled probe. Digital PCR chips were fully saturated (100% positive wells) at concentrations of 107 copies/µl to 104 copies/µl. At the 103 copies/ µl concentration, approximately 69% of the wells displayed successful amplification, with 10% and 1% successful amplification observed for 102 and 101 copies/µl concentrations, respectively. The optimal concentration for the NAP plasmids was between 102 and 101 copies/µl, having 95% confidence intervals (CI) values 10% and below (Table 3).
Final eluate from the total DNA extractions of NAP also had successful amplification for all concentrations. At concentrations of total DNA of 25 ng/µl and 2.5 ng/µl, chips were fully saturated (100% of wells reading as positive) while concentrations of 0.25 ng/µl and 0.025 ng/µl total DNA yielded approximately 31% and 3% positive wells. The calculated copy number for concentrations of 0.25 ng/µl and 0.025 ng/µl were 901.56 (21.7 95% CI) and 74.16 (5.52 95% CI), respectively (Table 3).