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.