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 .