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Reliable methods are needed to detect total and pathogenic Vibrio parahaemolyticus. One marker of V. parahaemolyticus virulence is the thermostable-related hemolysin. We developed an alkaline phosphatase–labeled DNA probe method for the specific detection and enumeration of trh-positive V. parahaemolyticus by colony hybridization. The probe was tested against a panel of 200 bacterial strains and determined to be specific for trh-positive V. parahaemolyticus. Additionally, the trh alkaline phosphatase probe colony hybridization was successfully used to detect and enumerate trh-positive V. parahaemolyticus in seafood and water samples collected from the United States and the United Kingdom.