A Simplified, CRISPR-Based Method for the Detection of Batrachochytrium salamandrivorans

The fungal pathogen Batrachochytrium salamandrivorans (Bsal) is one of two species (the other, B. dendrobatidis/Bd) that cause amphibian chytridiomycosis, an emerging infectious disease that has been indicated in the declines of hundreds of amphibian species worldwide. While Bd has been near globally distributed for decades, Bsal is a more recently emerged pathogen, having been identified just over a decade ago with current impacts localized to salamandrids in parts of Europe. However, because there is concern that Bsal will cause widespread declines if introduced to naïve regions-such as the Americas where the greatest diversity of salamandrids exist-it is imperative that widespread monitoring strategies be implemented to mitigate the spread of Bsal. As standard molecular diagnostic approaches-such as qPCR-tend to be expensive, time-consuming, or require specialized instrumentation and training, we have developed a simplified, rapid, CRISPR-based approach for Bsal-DNA detection. Here, we demonstrate this approach-termed FINDeM (Field-deployable, Isothermal, Nucleotide-based Detection Method)-and show that it can detect clinically relevant concentrations of Bsal DNA in under an hour using only inexpensive supplies and body-heat inducible reactions. Further, we highlight drawbacks and limitations associated with FINDeM-such as decreased DNA extraction yields and detection sensitivity when compared to more traditional approaches-and provide suggestions for additional development and future application of this method.