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Characterization of a Cloned pR72H Probe for Vibrio parahaemolyticus Detection and Development of a Nonisotopic Colony Hybridization Assay

Vibrio parahaemolyticus is a halophilic bacterium often found in shellfish and is an important causative agent of food poisoning in Taiwan. A rapid and efficient detection method is required to identify this foodborne pathogen. A 0.76‐Kb HindIII DNA fragment was cloned from the chromosomal DNA of V. parahaemolyticus strain no. 93, designated as pR72H fragment, was used as a polynucleotide probe. It was labeled with digoxigenin‐11‐dUTP (DIG) by the random primer‐labeling method. The sensitivity and specificity of the digoxigenin‐labeled 0.76‐Kb DNA probe was determined by colony hybridization assay. Under stringent hybridization conditions, 122 of 124 isolates of V. parahaemolyticus showed positive hybridization reaction with DIG‐0.76‐Kb DNA probe; the negative strains were attributed to slow growth. The DIG‐0.76‐Kb probe did not hybridize with 86 isolates of other vibrios and a number of other enterics as well as nonenteric microorganisms. The sensitivity and specificity of this DIG probe are 98% and 100%, respectively. This nonisotopic colony hybridization assay can be very useful for routine monitoring of V. parahaemolyticus in the food industry, environmental analysis and clinical laboratories.