DNA extraction and quantitative real-time PCR
Total eDNA on the filter was extracted using the DNeasy Blood and Tissue Kit (Qiagen, Germany), according to the method described by Jo et al. (2017). We estimated eDNA concentration in water samples by quantifying the copy number of CytB genes and ITS1 regions using the StepOnePlus Real-Time PCR system (Thermo Fisher Scientific, USA). In this study, we targeted four types of Japanese jack mackerel eDNA, short and long fragments of the mitochondrial cytochrome b (CytB) gene and nuclear internal transcribed spacer-1 (ITS1) region in the ribosomal RNA (rRNA) gene (mtS, mtL, nuS, and nuL, respectively) for the eDNA quantification. We cited primers/probe sets amplifying mtS, mtL, and nuS of Japanese jack mackerel eDNA from previous literature (Table 2), and newly developed the primers/probe sets that amplify 603 bp fragments of the ITS1 region in Japanese jack mackerel (Table S2; Appendix S1). Each 13.3 μL of TaqMan reaction contained 2 μL template DNA, a final 900 nM concentration of both the forward and reverse primers, and 125 nM of TaqMan probe in 1 × TaqPathTM qPCR Master Mix, CG. We simultaneously analyzed 2 μL of pure water as a negative PCR control. We performed qPCR using a dilution series of standards containing 3 × 101–3 × 104 copies of a linearized plasmid containing synthesized artificial DNA fragments from the CytB gene (1141 bp) or ITS1 region (666 bp) of target species. All eDNA samples, standards, and negative controls were performed in triplicates. The thermal conditions for qPCR were as follows: 2 min at 50 °C, 10 min at 95 °C, 55 cycles of 15 s at 95 °C and 1.5 min at 60 °C (2-step PCR) for mtS and nuS and 2 min at 50 °C, 10 min at 95 °C, 55 cycles of 15 s at 95 °C, 30 s at 60 °C, and 1 min at 72 °C (3-step PCR) for mtL and nuL. We calculated eDNA concentrations by averaging the triplicate, and each PCR-negative replicate (indicating non-detection) was regarded as containing zero copies (Ellison et al., 2006).