Model pc 808

Primer sets, expected to be specific to the Vibrio genus and/or species, were designed from each of the screened candidate genes and were evaluated using genomic DNAs of Vibrio and other type strains listed in Table 1. PCR amplifications were carried out with 200 μM of each dNTP, 0.5 unit of Ex Taq DNA polymerase (TaKaRa Bio Inc., Shiga, Japan), 1× Ex Taq buffer, 25 ng of template DNA and the adjusted concentration of each primer in a final reaction volume of 25 μl. PCR amplification was performed in a thermocycler (Model PC 808, ASTEC, Fukuoka, Japan) with an initial denaturation at 94 °C for 5 min, followed by 25 cycles of 94 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s, finishing with a final extension at 72 °C for 10 min and storage at 4 °C thereafter. Amplified products were electrophoresed on a 3 % agarose gel in 0.5× Tris-acetate-EDTA buffer, stained with ethidium bromide, visualized under UV-irradiation and photographed with a digital camera (Model COOLPIX 4300, Nikon, Tokyo, Japan).

Single PCR was conducted under the following conditions: the final volume was 25 µL, containing 2.5 µL 10× buffer (Bioneer, Daejeon, Korea), 0.2 mM dNTPs, 0.5 unit Hot Start Taq DNA polymerase (Bioneer), 0.4 µM of each primer, and 10 ng template DNA. Amplification was conducted in a thermal cycler (Model PC 808, ASTEC, Fukuoka, Japan) as follows: predenaturation at 95 °C for 5 min; 40 cycles of 95 °C for 30 s, 62 °C for 30 s, and 72 °C for 30 s; and a final extension at 72 °C for 5 min. The amplification products were electrophoresed on 2% agarose gel stained with ethidium bromide at 150 V for 12 min.
For multiplex PCR, the optimized concentration of primers (Table 1) and 1 unit of Hot Start Taq DNA polymerase were used, and the rest of the conditions were the same as single PCR. The amplification products from the multiplex PCR were electrophoresed on 3% agarose gel stained with ethidium bromide at 150 V for 30 min. Additionally, capillary electrophoresis was conducted using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) to confirm the sensitive accuracy of multiplex PCR.