tetano
Editor, Senior Moderator
J Virol Methods. 2015 Aug 24. pii: S0166-0934(15)00290-6. doi: 10.1016/j.jviromet.2015.08.014. [Epub ahead of print]
[h=1]Optimization of Droplet Digital PCR from RNA and DNA extracts with direct comparison to RT-qPCR: Clinical implications for quantification of Oseltamivir-resistant subpopulations.[/h] Taylor SC[SUP]1[/SUP], Carbonneau J[SUP]2[/SUP], Shelton DN[SUP]3[/SUP], Boivin G[SUP]2[/SUP].
[h=3]Author information[/h]
[h=3]Abstract[/h] 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 C[SUB]q[/SUB] and ΔC[SUB]q[/SUB] 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.
Copyright ? 2015 The Authors. Published by Elsevier B.V. All rights reserved.
[h=4]KEYWORDS:[/h] Droplet Digital PCR; Experimental optimization; Influenza virus; RT-qPCR; ddPCR
PMID: 26315318 [PubMed - as supplied by publisher] Free full text
[h=1]Optimization of Droplet Digital PCR from RNA and DNA extracts with direct comparison to RT-qPCR: Clinical implications for quantification of Oseltamivir-resistant subpopulations.[/h] Taylor SC[SUP]1[/SUP], Carbonneau J[SUP]2[/SUP], Shelton DN[SUP]3[/SUP], Boivin G[SUP]2[/SUP].
[h=3]Author information[/h]
[h=3]Abstract[/h] 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 C[SUB]q[/SUB] and ΔC[SUB]q[/SUB] 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.
Copyright ? 2015 The Authors. Published by Elsevier B.V. All rights reserved.
[h=4]KEYWORDS:[/h] Droplet Digital PCR; Experimental optimization; Influenza virus; RT-qPCR; ddPCR
PMID: 26315318 [PubMed - as supplied by publisher] Free full text