Optimization of Droplet Digital PCR from RNA and DNA extracts with direct comparison to RT-qPCR: Clinical implications for quantification of Oseltamivir-resistant subpopulations

Abstract

The recent introduction of Droplet Digital PCR (ddPCR) has provided researchers with a tool that permits direct quantification of nucleic acids from a wide range of samples with increased precision and sensitivity versus RT-qPCR. The sample interdependence of RT-qPCR stemming from the measurement of Cq and ΔCq values is eliminated with ddPCR which provides an independent measure of the absolute nucleic acid concentration for each sample without standard curves thereby reducing inter-well and inter-plate variability. Well-characterized RNA purified from H275-wild type (WT) and H275Y-point mutated (MUT) neuraminidase of influenza A (H1N1) pandemic 2009 virus was used to demonstrate a ddPCR optimization workflow to assure robust data for downstream analysis. The ddPCR reaction mix was also tested with RT-qPCR and gave excellent reaction efficiency (between 90% and 100%) with the optimized MUT/WT duplexed assay thus enabling the direct comparison of the two platforms from the same reaction mix and thermal cycling protocol. ddPCR gave a marked improvement in sensitivity (>30-fold) for mutation abundance using a mixture of purified MUT and WT RNA and increased precision (>10 fold, p<0.05 for both inter- and intra-assay variability) versus RT-qPCR from patient samples to accurately identify residual mutant viral population during recovery.

The Future of Digital Polymerase Chain Reaction in Virology

“…For CMV, Hayden et al [12] and Sedlak et al [47] found a higher precision at high CMV concentrations, although equal precision was found for lower levels of CMV. Similar findings have been reported for hepatitis C virus (HCV) [29], HBV [20] and influenza [30].…”

“…This dPCR approach may apply well in the setting of virology because emerging drug resistance mutations also need to be screened/monitored. In this context, a small number of papers have already explored this possibility, indicating that dPCR is more sensitive, compared with qPCR, at detecting low levels of emerging drug-resistant mutations [29][30][31]. Alternatively, dPCR can also be used for comparing the ratio of viral sequences to the number of haploid cellular genomes.…”

“…Both Whale et al [31] and Taylor et al [30] studied the sensitivity to detect influenza A mutants and found an increase in sensitivity of more than 50-and 30-fold, respectively. Such increased sensitivity, combined with the precision of dPCR, may allow a diagnostic application of dPCR, especially when specific mutations can be targeted by allele-specific PCR.…”

Droplet Digital PCR versus qPCR for gene expression analysis with low abundant targets: from variable nonsense to publication quality data

“…A single reaction mix was therefore produced for each sample and for all experiments which was split (20 µL each) for data acquisition between platforms (see Materials and Methods) as similarly designed in a previous study 13 . Since qPCR is a sample interdependent technology where the relative quantity and normalized gene expression data rely on ΔCq values, the analysis from a single plate assures the best quality results by eliminating any bias from inter-plate variability 16, 17 .…”

“…However, there are two distinct differences: 1) the partitioning of the PCR reaction into thousands of individual reaction vessels prior to amplification and 2) the acquisition of data at reaction end point. These factors offer the advantage of direct and independent quantification of DNA without standard curves giving more precise and reproducible data versus qPCR especially in the presence of sample contaminants that can partially inhibit Taq polymerase and/or primer annealing 11–13 . In addition, end-point measurement enables nucleic acid quantitation independently of the reaction efficiency, resulting in a positive-negative call for every droplet and greater amenability to multiplexed detection of target molecules 14 .…”