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Comparative Study of Sensitivity, Linearity, and Resistance to Inhibition of Digital and Nondigital Polymerase Chain Reaction and Loop Mediated Isothermal Amplification Assays for Quantification of Human Cytomegalovirus

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† LGC, Queens Road, Teddington, Middlesex TW11 0LY, United Kingdom
‡ Research Department of Infection, Division of Infection and Immunity, UCL, Gower Street, London WC1E 6BT, United Kingdom
§ Virology Laboratory, Clinical Microbiology and Virology, UCLH NHS Foundation Trust, 60 Whitfield Street, London W1T 4EU, United Kingdom
*E-mail: [email protected]. Fax: +44 (0)20 8943 2767.
Cite this: Anal. Chem. 2014, 86, 9, 4387–4394
Publication Date (Web):March 31, 2014
https://doi.org/10.1021/ac500208w
Copyright © 2014 American Chemical Society
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    Performing nucleic acid amplification techniques (NAATs) in digital format using limiting dilution provides potential advantages that have recently been demonstrated with digital polymerase chain reaction (dPCR). Key benefits that have been claimed are the ability to quantify nucleic acids without the need of an external calibrator and a greater resistance to inhibitors than real-time quantitative PCR (qPCR). In this study, we evaluated the performance of four NAATs, qPCR, dPCR, real-time quantitative loop mediated isothermal amplification (qLAMP), and digital LAMP (dLAMP), for the detection and quantification of human cytomegalovirus (hCMV). We used various DNA templates and inhibitors to compare the performance of these methods using a conventional real-time thermocycler platform (Bio-Rad CFX96) and a chip based digital platform (Fluidigm Biomark 12.765 Digital Array). dPCR performed well and demonstrated greater resistance to inhibitors than the other methods although this resistance did not apply equally to all inhibitors tested. dLAMP was found to be less sensitive than dPCR, but its quantitative performance was better than qLAMP, the latter being unable to quantify below 1000 copies. dLAMP was also more resistant to inhibitors than qLAMP. Unlike qPCR, both digital methods were able to quantify viral genomes without requiring a calibrator; however, neither can currently compete with the large reaction volumes, and thus the greater absolute sensitivity, of qPCR. With the introduction of digital instrumentation that will enable larger reaction volumes, digital amplification methods such as those evaluated in this study could potentially offer a robust alternative to qPCR for nucleic acid quantification.

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