Identification of Vibrio halioticoli by colony hybridization with non‐radioisotope labeled genomic DNA probe

Vibrio halioticoli was isolated from the gut of abalone Haliotis discus hannai and identified as a new member of the genus Vibrio. Vibrio halioticoli was first identified as a non-motile fermenter (NMF) because of its facultative anaerobic, alginolytic, and non-motile traits. Most of the gut isolates from abalone H. discus hannai were identified as NMF using phenotypic characterization, and V. halioticoli strains were found to be dominant in the gut. From an ecophysiological view point, NMF strains have an alginate-degrading ability, which probably degrades the polyguluronate block, and might assist in the breakdown of the alginate-rich microalgae ingested by abalone. In other words, a symbiotic relationship similar to that of ruminants may be found between NMF and abalone. We have developed a species-specific detection and identification method for V. halioticoli that is able to differentiate precisely and rapidly V. halioticoli strains from other NMF isolates. An applicable fingerprinting technique based on 16S rDNA polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) has been reported previously by Tanaka et al. V. halioticoli strains were differentiated from others using RFLP; however, as the PCR/RFLP technique is suitable only for phenotypically characterized isolates, cost and time restricts its application to huge amounts of isolates. In the present study, we developed a colony hybridization technique for the species-specific detection of V. halioticoli using alkaline phosphatase-labeled genomic DNA probes instead of conventional radioisotope probes. This method allows the rapid identification and enumeration of V. halioticoli strains grown on agar plates without phenotypic characterization and isolation. Vibrio halioticoli IAM14596 genomic DNA was purified according to Marmur, and the DNA concentration was adjusted 20 ng/mL in a 1.5 mL microcentrifuge tube. The DNA was denatured at 100∞C for 5 min, then transferred immediately on ice and kept for 1 h. The denatured DNA solution was randomly broken by 30 times sonication at 50 W for 5 min. The denatured and sonicated DNA fragments were labeled using the Alkphos direct labeling system (Amersham Pharmacia Biotech, Chicago, IL, USA) according to the instruction manual. The labeled mixture, containing heatstable alkaline phosphatase provided from the labeling system, was added to 10 mL of the aboveprepared DNA solution, and incubated at 37∞C for 30 min The V. halioticoli genomic DNA probes (Vh probes) labeled with alkaline phosphatase were stored at 4∞C before hybridization. The nylon membrane (Hybond-N, 82 mm diameter, Amersham Pharmacia Biotech) spotted template DNA or transferred colonies were placed onto Whatman 3MM paper that had been immersed previously in a denaturation buffer (0.5 M NaOH–1.5 M NaCl) for 5 min at room temperature, hence causing lysis of cells and the denaturation of genomic DNA. The membrane was placed briefly onto Whatman paper to remove any Short Paper