4.3. Enrichment, Cultivation, and Molecular Profiling of V. vulnificus

The pre-treated water sample and 1 mL shellfish homogenized liquid sample was transferred into 10 mL Alkaline Peptone Water (APW; Taiwan Prepared Media, Taipei, Taiwan), followed by incubation at 30 °C, for 24 h, for enrichment. The next day, a loopful of broth from the pre-enrichment step was streaked on CHROMagar Vibrio (CV; Taiwan Prepared Media, Taipei, Taiwan) plates and Thiosulfate Citrate Bile Salt Sucrose (TCBS; Taiwan Prepared Media, Taipei, Taiwan) agar plate, followed by incubating at 37 °C, for 24 h. The next day, a single colony from agar plate was picked with the help of a toothpick and inoculated into an APW tube and then incubated at 30 °C, for 24 h. After incubation, 300 µL culture containing candidate isolates were mixed with 700 µL 33% glycerol into a 1.5 mL centrifuge tube and stored at −20 °C. The reference strain source in this study was V. vulnificus ATCC27562, which served as a control for subsequent experimental analysis. The DNA extraction from overnight culture was centrifuged at 10,000× g for 5 min and removed from the supernatant. DNA from concentrated pellet was extracted for molecular analysis using ZP02006 MagPurix automatic DNA extraction system (Zinexts Life Science Corp, New Taipei, Taiwan) provided with the bacterial DNA extraction kit, following the procedure of the manufacturer’s instructions, with the final elution in 100 µL. This eluent was then used for PCR experiments with a total reaction volume of 25 µL by adding the appropriate concentration of template, primers, and master mix, respectively, as shown in Table 1. PCR amplification was performed using Life ECO Thermal Cycler (Bioer Technology Co., Ltd., Hangzhou, China) according to the conditions of the PCR programs, as shown in Table 4. Finally, DNA quality was assessed visually by running gel electrophoresis with 1.5% 1 × TAE Buffer at 110 V, for 30 min, and bands were visualized under UV. The positive samples were identified by targeting the vvhA gene in PCR, which is a common gene marker for the identification of V. vulnificus species. Subsequently, the detected five major virulence-associated genes, including V. vulnificus cytolysin⁄hemolysin gene (vvhA), viral correlated gene (vcg), capsular polysaccharide operon (CPS), pathogenicity region XII (PRXII), Sial acid catastrophe region (nanA), and enzyme IIA of mannitol fermentation operon (manIIA) were used for phylogenetic analysis and strain typing, by means of ERIC-PCR fingerprinting and with the aid of commercial software BioNumerics (Applied Maths NV, Inc., Sint-Martens-Latem, Belgium). Finally, the cluster analysis was performed using curve-based Pearson correlation, and the resulting dendrogram was generated based on the unweighted pair group method with arithmetic mean (UPGMA).

PCR primers and conditions for targeting toxin gene profiles, and the identification and differentiation of V. vulnificus.