title: 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.
authors: Bio-Rad Laboratories Canada, Inc., 1329 Meyerside Drive, Mississauga, ON, Canada L5T1C9. Electronic address: sean_taylor@bio-rad.com.
authors: Sean C
authors: SC
authors: Taylor
authors: CHU of Quebec and Laval University, Quebec City, QC, Canada.
authors: Julie
authors: J
authors: Carbonneau
authors: Digital Biology Center, Bio-Rad Laboratories, Pleasanton, CA, USA.
authors: Dawne N
authors: DN
authors: Shelton
authors: CHU of Quebec and Laval University, Quebec City, QC, Canada.
authors: Guy
authors: G
authors: Boivin
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology*; D002675:Child, Preschool; D024882:Drug Resistance, Viral; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000145:classification / Q000187:drug effects* / Q000235:genetics* / Q000302:isolation & purification; D007251:Influenza, Human / Q000821:virology; D008297:Male; D009439:Neuraminidase / Q000235:genetics; D053139:Oseltamivir / Q000494:pharmacology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019562:Viral Load / Q000379:methods*; D014764:Viral Proteins / Q000235:genetics
chemical_list: D000998:Antiviral Agents; D014764:Viral Proteins; D053139:Oseltamivir; C487630:NA protein, influenza A virus; D009439:Neuraminidase
keywords: Droplet Digital PCR; Experimental optimization; Influenza virus; RT-qPCR; ddPCR