Rapid and specific detection of the thermostable direct haemolysin gene in Vibrio parahaemolyticus by the polymerase chain reaction

Abstract

Synthetic oligonucleotide primers derived from a sequence of the thermostable direct haemolysin (tdh) gene were used in a polymerase chain reaction (PCR) amplification technique to detect this gene in strains of Vibrio parahaemolyticus. A total of 36 TDH-producing, and 89 TDH-negative Vibrio parahaemolyticus strains and 46 other vibrios and enteric pathogens were studied. In all, 36 strains of Vibrio parahaemolyticus from which the tdh gene could be successfully amplified by PCR were found to be TDH-positive in TDH haemolysin assay. No amplification products were obtained from Vibrio parahaemolyticus strains that were TDH-negative in the haemolysin assay or from other vibrios and enteric pathogens, with the exception of two strains. The PCR results were consistent with DNA hybridization tests. The detection limit for the tdh gene by PCR amplification was 40 pg of total DNA, or broth culture containing 1000 viable cells. Amplification products were confirmed by restriction enzyme digestion and Southern blot hybridization. The PCR method could detect the tdh sequences in stool samples from patients with gastroenteritis caused by V. parahaemolyticus. This PCR protocol clearly identified TDH-producing strains of V. parahaemolyticus and provides an alternative to conventional methods for TDH detection by research laboratories, clinical laboratories, regulatory agencies, and the seafood industry.