Quantitative PCR
For C. sivickisi , the RT-PCR analysis was based on a TaqMan
hydrolysis probe (Cop_siv_16S_P - 5’-AACTATTCGCCTCAC-3’) assay (Table
1). Each qPCR reaction was run on a QuantStudio 5 Real-Time PCR machine
(Thermo Fisher Scientific) and consisted of 5.0 μL 1x TaqPath, 0.9 μM
Copula_16S_F (Forward), 0.9 μM Copula_16S_R (Reverse), 0.25 μM
Copula_16S_P probe, 2.5 μL sample and 0.7 μL of MilliQ for a final
reaction volume of 10 μL. Results were obtained using optimised
thermocycling for 40 cycles for 5 min at 95° C, 30 s at 95° C, 15 s at
60° C.
For all other species (C. fleckeri, C. xaymacana and C.
barnesi ), eDNA detection was via a SYBR Green Power Up assay, with each
reaction consisting of 1.4 μL MilliQ, 10 μL 2x PowerUp SYBR Green, 0.9
μM Forward primer, 0.9 μM Reverse primer, 5 μL sample for a final
reaction volume of 20 μL. This reaction volume was chosen due to the
lower specificity when compared to the hydrolysis probe and ran at
optimised thermocycling for 50 cycles of 2 min at 50 °C, 2 min at 95 °C
, 15 s at 95 °C, 1 min at 65 °C, utilising a Melt Curve of 15 s at 95
°C, 1 min at 60 °C, 15 s at 95 °C. Each sample was run in triplicate.
Water samples that produced a PCR product were sent for Sanger
sequencing (AGRF, Brisbane, Australia) to confirm the
species-specificity of the resulting PCR product. For all species,
amplification of a single technical replicate deemed the sample
replicate as a positive detection.