Multiplex optimization
The multiplex assay was purchased directly from ThermoFisher. The 5’-ends of each species-specific probe were labeled accordingly; FAM-NAP, VIC-RB, ABY-BP and JUN-BC. All assays tagged on 3’ end with MGB-NFQ quencher.
Plasmid standards and samples for each snake species with the optimal concentrations were screened using the multiplex assay. In addition, samples with optimal concentrations for each snake species were mixed in all possible combinations (C(n,r)=n!/r!( n-r!)) to reflect the possibility of an eDNA sample containing multiple targets.
All reactions were run in volumes of 9 µl and comprised of 1.8 µl Absolute Q Master Mix (5x), 0.45 µl dPCR assay (20x), 1 µl DNA template and the remaining volume made up with UltraPure water. Thermal cycling conditions were as follows; 10 min. initial denaturation at 95 °C followed by 25 cycles of denaturation for 15 sec. at 95 °C and annealing/extension for 1 min. at 58 °C. All reactions were run on the QuantStudio™ Absolute Q™ Digital PCR System (ThermoFisher Scientific).
Results