CLONING AND NUCLEOTIDE-SEQUENCE OF THE GYRB GENE OF VIBRIO-PARAHAEMOLYTICUS AND ITS APPLICATION IN DETECTION OF THIS PATHOGEN IN SHRIMP

Because biochemical testing and 16S rRNA sequence analysis have proven inadequate for the differentiation of Vibrio parahaemolyticus from cl osely related species, we employed the gyrase B gene (gvrB) as a molec ular diagnostic probe. The gyrB genes of V. parahaemolyticus and close ly related Vibrio alginolyticus were cloned and sequenced. Oligonucleo tide PCR primers were designed for the amplification of a 285-bp fragm ent from within gyrB specific for V. parahaemolyticus. These primers r ecognized 117 of 117 reference and wild-type V. parahaemolyticus strai ns, whereas amplification did not occur when 90 strains of 37 other Vi brio species or 60 strains representing 34 different nonvibrio species were tested. In 100-mu l PCR mixtures, the lower detection limits wer e 5 CFU for live cells and ii pg for purified DNA. The possible applic ation of gyrB primers for the routine identification of V. parahaemoly ticus in food was examined. We developed and tested a procedure for th e specific detection of the target organism in shrimp consisting of an 18-h preenrichment followed by PCR amplification of the 285-bp V. par ahaemolyticus-specific fragment. This method enabled us to detect an i nitial inoculum of 1.5 CFU of V. papahaemolyticus cells per g of shrim p homogenate. By this approach, we were able to detect V. parahaemolyt icus in all of 27 shrimp samples artificially inoculated with this bac terium. We present here a rapid, reliable, and sensitive protocol for the detection of V. parahaemolyticus in shrimp.