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Diseases of Aquatic Organisms

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DAO 141:139-147 (2020)  -  DOI: https://doi.org/10.3354/dao03518

Choice of molecular assay determines ranavirus detection probability and inferences about prevalence and occurrence

Felicity J. Wynne1, Robert Puschendorf1, Mairi E. Knight1, Stephen J. Price2,3,*

1School of Biological and Marine Sciences, University of Plymouth, Drake Circus, Plymouth, Devon, PL4 8AA, UK
2UCL Genetics Institute, Gower Street, London, WC1E 6BT, UK
3Institute of Zoology, Zoological Society of London, Regent’s Park, London, NW1 4RY, UK
*Corresponding author:

ABSTRACT: Ranaviruses are emerging pathogens that can cause morbidity, mortality and population declines in ectothermic hosts; however, there is no standardized approach to diagnostics. Here, we compared the inter-assay variation and intra-assay precision among 2 commonly used quantitative PCRs (qPCRs), a conventional and a nested PCR assay (used as a gold standard), using laboratory-propagated ranavirus (FV3 and CMTV) and field-collected samples. A qPCR assay (‘Leung’) detected viral DNA in dilutions 2 orders of magnitude lower than other assays regardless of the viral lineage of the cultured isolate (FV3/CMTV). The second qPCR (‘Brunner’) was slightly more sensitive than the conventional PCR (‘Mao’ assay). For field samples, the Leung qPCR detected all known positives, while the Mao assay PCR only detected 2.5% of the positive samples. Amplicon sequences from the 2 conventional PCRs were shown to be useful for inferring viral lineage. Inaccurate results will bias estimates of the distribution and prevalence of ranaviruses, and together these findings emphasize that molecular assays should be chosen carefully in the context of study aims.


KEY WORDS: Ranavirus · Diagnostic methods · qPCR · Nested PCR · Specificity · Sensitivity


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Cite this article as: Wynne FJ, Puschendorf R, Knight ME, Price SJ (2020) Choice of molecular assay determines ranavirus detection probability and inferences about prevalence and occurrence. Dis Aquat Org 141:139-147. https://doi.org/10.3354/dao03518

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