Filed Protocol for DNA Analysis
We used reagents, assay, handheld qPCR and protocols from Biomeme Inc.
(Biomeme Inc, Philadelphia PA). We extracted all samples in the field
within 10 min of collection using M1 Sample Prep Kit and protocol for
eDNA or DNA respectively and we extracted DNA from the entire swab or
filter. We analyzed the samples using Go-Strips™, custom shelf-stable
assay developed from previously published Bd qPCR assay (Boyle et al.,
2004). The Go-Strips contain primer, probe, master mix, and internal
positive control and only require the addition of extracted DNA. The
exception to this was at site 1, where we added pre-mixed primer and
probe stored on ice to the Go-Strips that contained master mix and
internal positive control. We ran our samples on a two3™ mobile
real-time PCR machine (~ 2 lbs.). To quantify initial
DNA copy number of Bd in the samples, we created a standard curve by
using a three-point serial dilution (100-10,000 copies) of a synthesized
gene (gBlocks; Integrated DNA) prior to running reactions. We used 20-40
µl of DNA extract in each reaction (20 µl at site 1, and 40 µl at site 2
and 3). The two3™ contains three wells and can run two channels. We ran
each sample in triplicate (three technical replicates using all
available wells) with an internal positive control (duplex reaction
using both channels). As a result, we could not run a standard curves or
field negative simultaneously (as described in Sepulveda et al. 2018).
To run all skin swab reactions, we used a cycle of 15 min at 95°C
followed by 45 cycles at 94°C for 60 s and 60°C for 60 s. To run all
eDNA reactions, we used cycles of 15 min at 95°C followed by 50 cycles
at 94°C for 60 s and 60°C for 60 s. If inhibition occurred, we diluted
samples 1:1 using molecular grade water and re-ran. We diluted inhibited
samples in the field using a 20µl pipette. We analyzed the eDNA negative
control samples collected on site as the negative control for field PCR.