Multiplex validation and optimization
The multiplex assay successfully amplified for all reported dyes/assays for the corresponding snake species with no cross-amplification across snake species detected (Figure 2). For the BC plasmid (102 copies/µl) and sample (0.25 ng/µl) with a florescence threshold of 128.615, there were 21.7% and 29.7% positive wells detected for the JUN-BC probe, with zero positive wells for the ABY-BP, FAM-NAP and VIC-RB probes in both the plasmid and sample tests. For the BP plasmid (102 copies/µl) and sample (0.25 ng/µl) with a florescence threshold of 655.377, there were 9.6% and 31.41% positive wells detected for the ABY-BP probe, with zero positive wells for the JUN-BC, FAM-NAP and VIC-RB probes in both the plasmid and sample tests. For the NAP plasmid (102 copies/µl) and sample (0.25 ng/µl) with a florescence threshold of 61.813, there were 18.1% and 33.26% positive wells detected for the FAM-NAP probe, with zero positive wells for the JUN-BC, ABY-BP and VIC-RB probes in both the plasmid and sample tests. For the RB plasmid (102copies/µl) and sample (0.025 ng/µl) with a florescence threshold of 453.251, there were 37.6% and 11.29% positive wells detected for the VIC-RB probe, with zero positive wells for the JUN-BC, ABY-BP and FAM-NAP probes in both the plasmid and sample tests (Table 4).
All samples with mixed template screened with the multiplex assay displayed positive/negative patterns that directly corresponded to which species were present in the sample (Figure 3). Generally, the difference in efficiency and copy number estimate did not significantly change among two-species, three-species, and four-species mixtures. At low concentrations, the addition of additional template did not influence the assay. The 0.025 ng/µl concentration for RB, while yielding approximately 11 copies/µl by itself, consistently yielded significantly higher estimates in mixtures with other species. In addition, mixtures with RB template did display a reduction in copy number estimate as the amount of template increased, dropping from approximately 140 copies/µl in two-species combinations to approximately 70 copies/µl when mixed with all three other species.