Assay design and qPCR optimization
The initial PCR reactions on all four snake species using the universal primer set resulted in positive amplification. For each species, a 621 bp product was obtained. Sequences for each species were deposited in GenBank; B. constrictor = PP556870, E. cenchria = PP556871, P. sebae = PP556869 and P. bivittatus = PP556868 (Table 1). Resulting primers and probes for each respective snake species are presented in Table 1 along with optimal annealing temperatures and times. All four assays were found to run optimally at 58 °C for 1 min based on the gradient PCR. When run by qPCR with the TaqMan probe included, all assays were successful in amplifying all three replicates of the target species without cross amplification of non-target species (Table 2). Therefore, the BC assay successfully amplified from all three replicates of the B. constrictor sample but failed to amplify the target for E. cenchria , P. sebaeand P. bivittatus . The RB assay successfully amplified from all three replicates of the E. cenchria sample but failed to amplify the target for B. constrictor , P. sebae and P. bivittatus . The NAP assay successfully amplified from all replicates of the P. sebae sample but failed to amplify the target for B. constrictor , E. cenchria and P. bivittatus . Finally, the BP assay successfully amplified from all replicates of the P. bivittatus sample but failed to amplify the target for B. constrictor , E. cenchria and P. sebae .