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

Natalie K. Stilwell, Richard J. Whittington, Paul M. Hick, Joy A. Becker, Ellen Ariel, Steven Van Beurden, Niccolo Vendramin, Niels J. Olesen, Thomas B. Waltzek*

*Corresponding author for this work

    Research output: Contribution to journalJournal articleResearchpeer-review

    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.
    Original languageEnglish
    JournalDiseases of Aquatic Organisms
    Volume128
    Issue number2
    Pages (from-to)105-116
    ISSN0177-5103
    DOIs
    Publication statusPublished - 2018

    Keywords

    • Diagnostics
    • Quantitative PCR
    • Ranavirus
    • Sensitivity
    • Specificity

    Cite this

    Stilwell, N. K., Whittington, R. J., Hick, P. M., Becker, J. A., Ariel, E., Van Beurden, S., Vendramin, N., Olesen, N. J., & Waltzek, T. B. (2018). Partial validation of a TaqMan real-time quantitative PCR for the detection of ranaviruses. Diseases of Aquatic Organisms, 128(2), 105-116. https://doi.org/10.3354/dao03214