Figure Legend
Figure 1. Representative specimens of the species used to design the tetraplex digital PCR assay in this study; A) Burmese python (Python bivittatus ), B) North African rock python (Python sebae ), C) rainbow boa (Epicrates cenchria ) and D) boa constrictor (Boa constrictor ).
Figure 2. Chip images for the multiplex assay when screened against all four snake species (x-axis) with reporters/probes (y-axis) showing positive amplification for their corresponding species; red pixels = positive JUN florescence, yellow pixels = positive ABY florescence, green pixels = positive VIC florescence, blue pixels = positive FAM florescence, black pixels = no florescence/negative wells.
Figure 3. Absolute quantification of target DNA for each snake species when screened with tetraplex assay in mixed samples representing all possible combinations of multiple species present in a single sample.
Figure 4. Established populations of Burmese pythons (green), northern African pythons (blue), boa constrictors (purple), and rainbow boas (yellow) are shown for Miami-Dade County (blue outline) along with areas of range overlap among these species. Polygons representing each constrictor species were created using data available from EDDMapS (2024) using ArcGIS Pro, Version 3.2 and represent minimum bounding geometry.
Table 1. Primers and probes used for generating COI sequences and running qPCR and dPCR assays for optimization and developing the multiplex assay with corresponding critical PCR parameters.