2.3. DNA extraction and polymerase chain reaction (PCR) confirmation of E. ictaluri
Genomic DNA of all bacterial isolates (n = 26) was extracted using the InstaGene Matrix kit (Bio-Rad, California, USA). PCR tests were performed using genus- and species-specific primers targeting the fimbrial gene of E. ictaluri (generating 848 and 470 bp amplicons, respectively), as previously described (Sakai et al., 2009) (Table 2). Genomic DNA of E. ictaluri LMG7860 from striped catfish (purchased from BCCM/LMG Bacteria Collection, Gent, Belgium) was used as a positive control and nuclease-free water was used as a negative control. PCR reaction mixtures (20 µL) included 10 µL Gotaq Green Master Mix (Promega, Wisconsin, USA), 1.5 µL (10 µM) of the specific primer (forward and reverse), 3 µL DNA template, and 4 µL DNA-free distilled water. The mixtures were then placed in a thermal cycler for amplification under the following conditions: initial denaturation for 4 min at 94 °C; 35 cycles consisting of denaturation at 95 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 60 s; and a final extension for 7 min at 72 °C. The amplified products were then analyzed by electrophoresis on a 1% agarose gel containing a RedSafe nucleic acid staining solution (Intron, Gyeonggi-do, Korea). The images were digitally captured using a gel image system (Bio-Rad, California, USA).