Target gene for the assay was the V3-V4 hypervariable region of the 16S rRNA bacterial gene, defined by a 174 bp fragment using the following primers 341f 5 ′ -CCTACGGGAGGCAGCAG-3 ′ and 515r 5 ′ -ATTCCGCGGCTGGCA-3 ′ as described in López-Gutiérrez et al. (2004) (link). The Atlantic salmon elongation factor gene EL1-a (ELF) described in Bland et al. (2012) (link) was used as the reference gene in this assay, amplifying a 66 bp fragment using the primer set S-ELF.f 5'-GGCCAGATCTCCCAGGGCTAT-3' and S-ELF.r 5'-TGAACTTGCAGGCGATGTGA-3'.
Extracted DNA was diluted to a working concentration of 10 ng.µl for all samples. Real time PCRs were carried out in a ViiA TM 7 Real-Time PCR Machine (Applied Biosystems). qPCR was performed in a single-plex 25ul reaction containing 1.25 µl of 10 uM forward and reverse primer, 8 µl of RNase-free H 2 O, and 12.5 µl of SYBR Green qPCR Mastermix including hotstart Taq polymerase (Bioline). Each reaction contained 2 µl of normalized template DNA (30 ng.ul -1 ). PCR reactions were subjected to the following thermal cycling: 95
Extracted DNA was diluted to a working concentration of 10 ng.µl for all samples. Real time PCRs were carried out in a ViiA TM 7 Real-Time PCR Machine (Applied Biosystems). qPCR was performed in a single-plex 25ul reaction containing 1.25 µl of 10 uM forward and reverse primer, 8 µl of RNase-free H 2 O, and 12.5 µl of SYBR Green qPCR Mastermix including hotstart Taq polymerase (Bioline). Each reaction contained 2 µl of normalized template DNA (30 ng.ul -1 ). PCR reactions were subjected to the following thermal cycling: 95