DNA extraction
Each of the 1-L water samples was filtered immediately through a GF/F
glass fibre filter (normal pore size = 0.7 μm; diameter = 47 mm; GE
Healthcare Japan Corporation, Tokyo, Japan). To prevent
cross-contamination among the water samples, filter funnels and
measuring cups were bleached after filtration. All filters were
separately stored at -20°C until DNA extraction. Total eDNA was
extracted from each filter using a DNeasy blood and Tissue Kit (QIAGEN,
Hilden, Germany) and Salivette tubes (Sarstedt AG & Co. KG, Nümbrecht,
Germany). Extraction methods were performed according to Uchii, Doi, and
Minamoto (2016) with modifications. A filter sample was placed in the
upper part of the Salivette tube and 220 μL of solution containing 200
μL Buffer AL and 20 μL Proteinase K was added, and the tube containing
the filter was incubated at 56°C for 30 min. After that, the tube was
centrifuged at 5000 × g for 3 min, and the solution was collected
in the bottom part of the tube. To increase eDNA yield, 220 μL Tris-EDTA
(TE) buffer was added to the filter sample and re-centrifuged at 5000 ×g for 1 min. Then, 200 μL of ethanol was added to the collected
solution, and the mixture was transferred to a spin column. Then, total
eDNA was eluted in 100 μL of buffer AE following the manufacturer’s
instructions. All eDNA samples were stored at -20°C until qSeq and dPCR
were done.