Abi taqman environmental master mix 2

The eDNA was measured by quantitative PCR (qPCR) as described in a previous study^{5 (link)}, with slight modification. Instead of using ABI TaqMan Environmental Master Mix 2.0 (Applied Biosystems, Foster City, USA), Takara Probe qPCR Mix (Takara Bio Incorporation, Kusatsu, Japan) was used as we showed that this reagent has a higher capacity to resist inhibition by environmental contaminants in the PCR reaction (see Results). We compared the resistance to contaminants by Environmental Master Mix and Takara Probe qPCR Mix according to the spiking/dilution methods described previously^{5 (link)}, using the environmental samples collected from Murohama Bay of Otsuchi, which contained high levels of PCR inhibitors.

Laboratory analyses followed USEPA’s Method 1609 [22 ] and 1609.1 [23 ] in 2015 and 2016, respectively, with one modification: the salmon DNA sample processing control was used, but the plasmid internal amplification control was not. Briefly, the process included filtration of 100 mL through 47 mm polycarbonate filters with a 0.4 μm pore size. Following filtration, genomic DNA was extracted from the filters by bead-beating using pre-filled glass bead tubes (GeneRite, North Brunswick, NJ). In 2015 final crude genomic DNA extracts were diluted 5-fold in AE buffer and 5 μl were added, in duplicate, to 20 μl of ABI TaqMan® Environmental Master Mix 2.0 (Applied Biosystems, Foster City, CA), which contained the environmental master mix, primers, probes, bovine serum albumin, and nuclease-free water. Because of the low rate of inhibition observed in 2015 (described in detail in ‘Results’), in 2016 dilution of DNA extracts was not performed. Primers and probes (Integrated DNA Technologies, Inc., Coralville, Iowa) were as specified in Method 1609. All reactions were performed on the ABI StepOne Plus® platform.