Rapid detection of the amphibian chytrid fungus (Batrachochytrium
dendrobatidis) using in-situ DNA extraction and a handheld mobile
thermocycler
Abstract
The amphibian chytrid fungus (Bd) has caused extinction of amphibian
populations worldwide. Early and accurate Bd detection is essential for
management and treatment of susceptible anurans. We analyzed the
effectiveness of an in-situ DNA extraction along with handheld mobile
quantitative PCR (qPCR) thermocycler to detect Bd on skin frog swabs,
and to detect Bd 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 across all
three sites using both the field and lab protocols. We also detected Bd
DNA in eDNA samples collected at two of the three sites using both the
field and lab protocols although the field and lab protocol failed to
detect Bd eDNA at separate sites. The probability of detecting Bd DNA in
the technical replicates was lower for the field protocol compared to
samples extracted using 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 minutes. However, the applied field protocol may be prone
to false negatives when analyzing low-quantity DNA samples (i.e. eDNA).