Comparison of Droplet Digital PCR to Real-Time PCR for Quantitative Detection of Cytomegalovirus

Departments of Pathology.
Journal of clinical microbiology (Impact Factor: 3.99). 12/2012; 51(2). DOI: 10.1128/JCM.02620-12
Source: PubMed

ABSTRACT Quantitative real-time PCR has been widely implemented for clinical viral load testing, but a lack of standardization and relatively poor precision has hindered its usefulness. Digital PCR offers highly precise, direct quantification without requiring a calibration curve. Performance characteristics of real-time PCR were compared to those of droplet digital PCR (ddPCR) for cytomegalovirus (CMV) viral load testing. Ten-fold serial dilutions of the World Health Organization (WHO) and the National Institute of Standards and Technology (NIST) CMV quantitative standards were tested, together with the AcroMetrix® CMV tc Panel (Life Technologies, Carlsbad, CA) and 50 human plasma specimens. Each method was evaluated using all three standards for quantitative linearity, lower limit of detection (LOD), and accuracy. Quantitative correlation, mean viral load, and variability were compared. Real-time PCR showed somewhat higher sensitivity than ddPCR (LOD of 3 log(10)versus 4 log(10)copies and IU/mL for NIST and WHO standards). Both methods showed a high degree of linearity and quantitative correlation, for standards (R(2)≥ 0.98 in each of 6 regression models) and clinical samples (R(2)=0.93) across their detectable ranges. For higher concentrations, ddPCR showed less variability than RT-PCR for the WHO standards and Acrometrix standards (p< 0.05). RT-PCR showed less variability and greater sensitivity than did ddPCR in clinical samples. Both digital and real-time PCR provide accurate CMV viral load data over a wide linear dynamic range. Digital PCR may provide an opportunity to reduce quantitative variability currently seen using real-time PCR, but methods need to be further optimized to match the sensitivity of real-time PCR.

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Available from: Jessica Ingersoll, Oct 23, 2014
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    • "Although there are now hundreds of publications that underline the benefits of ddPCR, there have been reports concluding that RT-qPCR surpasses ddPCR in sensitivity and precision for clinical samples (Hayden et al., 2013). Although the Hayden study used controlled samples, the reaction conditions between ddPCR and RT-qPCR were much different in terms of cycling protocols, reaction mixes and total volumes which may account for this discrepancy as noted by the authors. "
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    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. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.
    Journal of virological methods 08/2015; DOI:10.1016/j.jviromet.2015.08.014 · 1.78 Impact Factor
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    • "reliance on rate-based measurements (C T values) and the need for the use of calibration curves (Hindson et al., 2011; Pinheiro et al., 2012). (2) It demonstrates high sensitivity and precision for lowcopy-number target nucleic acids (Diehl and Diaz, 2007; Hayden et al., 2013; Hindson et al., 2011; Sanders et al., 2011; Whale et al., 2012). In recent years, many studies (Jahn et al., 2014; McDermott et al., 2013; Nathan et al., 2014; Pinheiro et al., 2012) involving nucleic acid quantification have been performed using a commercial ddPCR system such as the Bio-rad QX100/200 (Sykes et al., 1992; Vogelstein and Kinzler, 1999). "
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    ABSTRACT: Sensitive and rapid identification of pathogenic bacterial is extremely important due to the serious threat of pathogens to human health. In this study, we demonstrate the simultaneous and sensitive detection of pathogenic Escherichia coli O157 and Listeria monocytogenes using a novel duplex droplet digital PCR (ddPCR) platform. The ddPCR platform, which uses a mineral oil-saturated polydimethylsiloxane (OSP) chip to overcome the problem of droplet evaporation, integrates the functions of droplet generation, on-chip amplification and end-point fluorescence readout. Simultaneous detection of two kinds of bacterial is achieved by the design of differentially labeled TaqMan-MGB fluorescent probes. Compared with a quantitative real-time PCR approach, the OSP chip-based duplex ddPCR platform exhibits high sensitivity, which is at the level of single molecule resolution without significant cross-assay interference. Moreover, the applicability of the proposed method is also evaluated in artificially contaminated drinking water sample, which displays a low detection limit down to 10CFU/mL for both pathogenic bacterial within 2h. Copyright © 2015. Published by Elsevier B.V.
    Biosensors & Bioelectronics 07/2015; 74:770-777. DOI:10.1016/j.bios.2015.07.016 · 6.41 Impact Factor
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    • "72 qPCR was performed using an Applied Biosystems 7300 system as 73 previously described [9]. dd-PCR was used in order to determine 74 the concentrations of the external DNA calibrators with multiple 75 probe sites [9] for qPCR because it accurately provides absolute 76 quantification of target DNA [3] [4] [6]. "
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    ABSTRACT: Two different quantitative PCR platforms, droplet digital PCR (dd-PCR) and quantitative real-time PCR (qPCR), were compared in a mcrA-based methanogen community assay that quantifies ten methanogen sub-groups. Both technologies exhibited similar PCR efficiencies over at least four orders of magnitude and the same lower limits of detection (8 copies μL-DNA extract−1). The mcrA-based methanogen communities in three full-scale anaerobic digesters were examined using the two technologies. dd-PCR detected seven groups from the digesters, while qPCR did five groups, indicating that dd-PCR is more sensitive for DNA quantification. Linear regression showed quantitative agreements between both of the technologies (R2 = 0.59–0.98) in the five groups that were concurrently detected. Principal component analysis from the two datasets consistently indicated a substantial difference in the community composition among the digesters and revealed similar levels of differentiation among the communities. The combined results suggest that dd-PCR is more promising for examining methanogenic archaeal communities in biotechnological processes.
    Biotechnology Reports 12/2014; 4. DOI:10.1016/j.btre.2014.06.010
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