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).