Lab Protocol for DNA Analysis
We stored the filters and swabs in 95% ethanol at room temperature away from any light source (Minamoto et al. 2016) and extracted the samples at Washington State University lab within 6 mo. following collection. For eDNA filters, we cut each filter in half, used half the filter for DNA extraction, and stored the other half in 95% ethanol as a reserved. We used a QIAshredder/Qiagen DNeasy Blood and Tissue DNA extraction protocol (Goldberg et al., 2011) in a limited-access room using best practices for eDNA (Goldberg et al., 2016). For frog swabs, we extracted DNA from the entire swab using Qiagen DNeasy Blood and Tissue DNA extraction protocol without QIAshredder in a tissue lab. We analyzed the samples using previously published Bd qPCR assay (Boyle et al., 2004), with the substitution of Environmental Master Mix (ThermoFisher, Waltham, MA), 3 µl of DNA extract in triplicate reactions, and running for 50 cycles, on a BioRad quantitative PCR (qPCR) machine (BioRad Laboratories, Hercules, CA). To quantify initial DNA copy number of Bd in the eDNA and swab samples, we created a standard curve by using a four-point serial dilution (10-10,000 copies) of a synthesized gene (gBlocks; Integrated DNA) in duplicate on each plate. We can detect quantities of DNA outside the range set by the standard curve, but exact quantities cannot be determined if they are outside the set range. All wells included an exogenous positive control to ensure no qPCR inhibition had occurred (IPC; ThermoFisher). We created and analyzed negative extraction and qPCR controls with every extraction batch and plate.