qPCR runs on field samples

I ran remaining field samples over the past 3 days or so. qPCR conditions were identical to those reported earlier except that cycle count was increased to 50 (plate 4) and 55 (plate 5). Plate configurations and results were as follows:

 

Plate 4 3/27/21
1 2 3 4 5 6 7 8 9 10 11 12
A H20 H20 H20 S2 S2 S2 Swi0m Swi0m Swi0m Swi0m Swi0m Swi0m
B S3 S3 S3 S4 S4 S4 Ava-S Ava-S Ava-S Ava-S Ava-S Ava-S
C S5 S5 S5 S6 S6 S6 Ava2m Ava2m Ava2m Ava2m Ava2m Ava2m
D S7 S7 S7 S8 S8 S8 Ava0m Ava0m Ava0m Ava0m Ava0m Ava0m
E Gol2m Gol2m Gol2m Gol2m Gol2m Gol2m RS311 RS311 RS311 RS311 RS311 RS311
F Gol-S Gol-S Gol-S Gol-S Gol-S Gol-S Hus-S Hus-S Hus-S Hus-S Hus-S Hus-S
G Swi-S Swi-S Swi-S Swi-S Swi-S Swi-S Hus2m Hus2m Hus2m Hus2m Hus2m Hus2m
H Swi2m Swi2m Swi2m Swi2m Swi2m Swi2m Hus0m Hus0m Hus0m Hus0m Hus0m Hus0m

 

Plate 5 3/29/21
1 2 3 4 5 6 7 8 9 10 11 12
A H20 H20 H20 S2 S2 S2 Sm210 Sm210 Sm210 Sm210 Sm210 Sm210
B S3 S3 S3 S4 S4 S4 Inf-S Inf-S Inf-S Inf-S Inf-S Inf-S
C S5 S5 S5 S6 S6 S6 Inf2m Inf2m Inf2m Inf2m Inf2m Inf2m
D S7 S7 S7 S8 S8 S8 Inf0m Inf0m Inf0m Inf0m Inf0m Inf0m
E Sla-S Sla-S Sla-S Sla-S Sla-S Sla-S Dra0m Dra0m Dra0m Dra0m Dra0m Dra0m
F Sla2m Sla2m Sla2m Sla2m Sla2m Sla2m Om0m Om0m Om0m Om0m Om0m Om0m
G Sla0m Sla0m Sla0m Sla0m Sla0m Sla0m Bay0m Bay0m Bay0m Bay0m Bay0m Bay0m
H RS029 RS029 RS029 RS029 RS029 RS029 Ava0m Ava0m Ava0m Ava0m Ava0m Ava0m

To summarize, the only field site where eDNA was detected in these two qPCR runs was a single replicate of Baytown 0m (Bay0m). This is notable because this is one of the four sites/samples that were re-run. The previous run did not detect any eDNA in Baytown 0m. This is likely to be simply a chance amplification of eDNA occurring at low abundance in the DNA extract; in other words, with enough replicates, many of these sites would probably show positive results. This argues for measures such as (1) increasing water filtrate volumes, (2) concentrating DNA extracts,  and/or (3) increasing template concentrations in the qPCR. I think options 1 and 3 make the most sense and suggest increasing filtrate volumes from 1L to 2L and perhaps doubling qPCR reaction volumes from 15 μl to 30 μl.