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).