DAO 128:105-116 (2018)  -  DOI: https://doi.org/10.3354/dao03214

Partial validation of a TaqMan real-time quantitative PCR for the detection of ranaviruses

Natalie K. Stilwell1,7, Richard J. Whittington2, Paul M. Hick2, Joy A. Becker3, Ellen Ariel4, Steven van Beurden5, Niccolò Vendramin6, Niels J. Olesen6, Thomas B. Waltzek1,*

1Department of Infectious Diseases and Pathology, College of Veterinary Medicine, University of Florida, Gainesville, FL 32610, USA
2Sydney School of Veterinary Science, Faculty of Veterinary Science, University of Sydney, 425 Werombi Rd, Camden, New South Wales 2570, Australia
3Sydney School of Life and Environmental Sciences, Faculty of Science, University of Sydney, 425 Werombi Road, Camden, NSW 2570, Australia
4College of Public Health, Medical and Veterinary Sciences, James Cook University, Townsville, Queensland 4811, Australia
5Department of Pathobiology, Utrecht University, 3508 TD Utrecht, The Netherlands
6Technical University of Denmark, National Veterinary Institute, Frederiksberg C, Denmark
7Present address: Seastar Communications and Consulting, Athens, GA 30606, USA
*Corresponding author:

ABSTRACT: Ranaviruses are globally emerging pathogens negatively impacting wild and cultured fish, amphibians, and reptiles. Although conventional and diagnostic real-time PCR (qPCR) assays have been developed to detect ranaviruses, these assays often have not been tested against the known diversity of ranaviruses. Here we report the development and partial validation of a TaqMan real-time qPCR assay. The primers and TaqMan probe targeted a conserved region of the major capsid protein (MCP) gene. A series of experiments using a 10-fold dilution series of Frog virus 3 (FV3) MCP plasmid DNA revealed linearity over a range of 7 orders of magnitude (107-101), a mean correlation coefficient (R2) of >0.99, and a mean efficiency of 96%. The coefficient of variation of intra- and inter-assay variability ranged from <0.1-3.5% and from 1.1-2.3%, respectively. The analytical sensitivity was determined to be 10 plasmid copies of FV3 DNA. The qPCR assay detected a panel of 33 different ranaviral isolates originating from fish, amphibian, and reptile hosts from all continents excluding Africa and Antarctica, thereby representing the global diversity of ranaviruses. The assay did not amplify highly divergent ranaviruses, members of other iridovirus genera, or members of the alloherpesvirus genus Cyprinivirus. DNA from fish tissue homogenates previously determined to be positive or negative for the ranavirus Epizootic hematopoietic necrosis virus by virus isolation demonstrated a diagnostic sensitivity of 95% and a diagnostic specificity of 100%. The reported qPCR assay provides an improved expedient diagnostic tool and can be used to elucidate important aspects of ranaviral pathogenesis and epidemiology in clinically and sublinically affected fish, amphibians, and reptiles.


KEY WORDS: Ranavirus · Quantitative PCR · Diagnostics · Sensitivity · Specificity


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Cite this article as: Stilwell NK, Whittington RJ, Hick PM, Becker JA and others (2018) Partial validation of a TaqMan real-time quantitative PCR for the detection of ranaviruses. Dis Aquat Org 128:105-116. https://doi.org/10.3354/dao03214

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