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Diseases of Aquatic Organisms

    DAO prepress abstract   -  DOI: https://doi.org/10.3354/dao03708

    Rapid detection of amphibian chytrid fungus Batrachochytrium dendrobatidis using in-situ DNA extraction and a handheld mobile thermocycler

    Colleen Kamoroff*, Caren S. Goldberg, Robert L. Grasso

    *Corresponding author:

    ABSTRACT: The amphibian chytrid fungus (Bd) has caused declines and some extinctions of amphibian populations worldwide. Early and accurate Bd detection is essential for management of susceptible anurans. We analyzed the effectiveness of an in-situ DNA extraction with a handheld mobile quantitative PCR (qPCR) thermocycler to detect Bd on frog skin swabs and in water samples using environmental DNA (eDNA). We collected duplicate eDNA samples and skin swabs from three Bd positive Rana sierrae populations. We processed one set of samples using a field protocol (a handheld thermocycler), and the other half using a standard lab protocol. We detected Bd DNA in all R. sierrae swabbed using both the field and lab protocols. We also detected Bd DNA in eDNA samples at all sites, although the field and lab protocol failed to detect Bd eDNA at separate singular sites; results from the field and lab eDNA protocol did not match. The probability of detecting Bd DNA in the technical replicates was lower for the field protocol compared to the lab protocol, suggesting the field protocol has reduced sensitivity and may not detect low quantities of DNA. Our results suggest the field extraction protocol using a handheld qPCR platform is a promising tool for rapid detection of Bd in susceptible amphibian populations. The field protocol yielded accurate results in less than 60 min. However, the applied field protocol may be prone to false negatives when analyzing low-quantity DNA samples such as eDNA water samples or frog swabs with low pathogen loads.