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