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Diseases of Aquatic Organisms

    DAO prepress abstract   -  DOI: https://doi.org/10.3354/dao03518

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

    Felicity J. Wynne, Robert Puschendorf, Mairi E. Knight, Stephen J. Price*

    *Corresponding author:

    ABSTRACT: Ranavirus is an emerging pathogen 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 two commonly used quantitative PCRs, a conventional and a nested PCR assay (used as a gold standard), using laboratory propagated ranavirus (FV3 & CMTV) and field-collected samples. A qPCR assay (‘Leung’) detected viral DNA in dilutions two 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 detecting 2.5% of the positive samples. Amplicon sequences from the two 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.