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


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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    • "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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