eDNA extraction and quantitative real-time PCR (qPCR)
We extracted total eDNA on the filter using a DNeasy Blood and Tissue
Kit (Qiagen, Germany), according to the method described in Minamoto et
al. (2019). The zebrafish eDNA concentration in the water sample was
estimated by quantifying the copy number of mitochondrial genes using
the StepOnePlus Real-Time PCR system (Thermo Fisher Scientific, U.S.).
We used the primer/probe sets to amplify the different lengths of
mitochondrial genes, including cytochrome b, tRNA-Glu, and ND6 regions
of target species (132, 430, 715, and 1021 bp) developed by Hirohara et
al. (2021). Each 15 μL of TaqMan reaction mixture contained a 2 μL DNA
template, a final 900 nM concentration of each forward and reverse
primer, and 125 nM of TaqMan probe in 1 × TaqPath qPCR Master Mix, CG
(Thermo Fisher Scientific). Simultaneously, 2 μL of pure water samples
were analyzed as the PCR negative controls. Target eDNA concentrations
were quantified based on a dilution series of standards containing 3 ×
101 to 3 × 104 copies of synthesized
artificial DNA fragments from zebrafish mitochondrial genes (Hirohara et
al., 2021). All qPCRs were performed in triplicate, including eDNA
samples, standards, and negative controls. Thermal conditions of the
qPCRs were 2 min at 50°C, 10 min at 95°C, 55 cycles of 15 s at 95°C, and
1 min (132 bp) or 1.5 min (other fragment lengths) at 60°C (Hirohara et
al., 2021). We calculated the eDNA concentrations by averaging the
triplicates, and each PCR negative replicate (indicating
non-amplification) was denoted as containing zero copies (Ellison et
al., 2006).