Enumeration and differentiation of Vibrio parahaemolyticus and Vibrio vulnificus by DNA-DNA colony hybridization using the hydrophobic grid membrane filtration technique for isolation

We have developed a means of differentiating and enumerating Vibrio parahaemolyticus and Vibrio vulnificus by DNA-DNA colony hybridization directly on HGMF filters. V. parahaemolyticus can be detected by a tdh-3-radiolabeled gene probe and V. vulnificus detected by a specific cytotoxin-hemolysin-radiolabeled probe with enumeration directly from autoradiograms. This procedure is more rapid than current techniques allowing enumeration and identification of these two species in samples as diverse as seawater, oyster (Crassostrea gigas), and shrimp (Pandalidae family) within 4 d. Our method is based on a rapid technique (18 h) for isolation and enumeration of V. parahaemolyticus from food using a membrane filtration technique with hydrophobic grid filters (HGMF). With the HGMF method, however, it is not possible to differentiate V. parahaemolyticus from V. vulnificus since on the HGMF-sucrose-based agar used, the two species are indistinguishable as both species are unable to ferment sucrose. Using a combination of the HGMF and selective gene probes, these two species can be differentiated