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 Vibvio pavahaemolyticus. A total of 36 TDH-producing, and 89 TDH-negative Vibvio pavahaemolyticus strains and 46 other vibrios and enteric pathogens were studied. In all, 36 strains of Vibrio pavahaemolyticus 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 Vibvio pavahaemolyticus 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. pavahaemolyticus. This PCR protocol clearly identified TDHproducing strains of V. pavahaemolyticus and provides an alternative to conventional methods for TDH detection by research laboratories, clinical laboratories, regulatory agencies, and the seafood industry.

Characterization of Vibrio Parahaemolyticus isolated from oysters and mussels in São Paulo, Brazil

“…Many studies have demonstrated that virulent strains of V. parahaemolyticus possess either the gene tdh or trh, or both 21 . The evidence of transmission of these genes through plasmids or insertion elements has already been demonstrated.…”

Application of polymerase chain reaction for detection ofVibrio parahaemolyticusassociated with tropical seafoods and coastal environment

“…Polymerase chain reaction (PCR) methods to detect the presence of these genes have been developed (Tada et al . 1992; Lee and Pan 1993; Karunasagar et al . 1996).…”

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

“…Total DNAs from 11 different V parahaemolyticus strains were isolated and digested with EcoRI, and Southern blots were prepared by using alkaline transfer. All hybridizations were performed under stringent conditions at 65 C. The hybridization results showed that all of the strains had an EcoRI fragment of about [13][14][15][16][17][18] Kb that hybridized with the pR72H probe (Fig. 2).…”