Detection of pathogenic Vibrio parahaemolyticus in oyster enrichments by real time PCR

Abstract

A real time polymerase chain reaction (PCR) assay was developed and evaluated to detect the presence of the thermostable direct hemolysin gene ( tdh), a current marker of pathogenicity in Vibrio parahaemolyticus. The real time PCR fluorogenic probe and primer set was tested against a panel of numerous strains from 13 different bacterial species. Only V. parahaemolyticus strains possessing the tdh gene generated a fluorescent signal, and no cross-reaction was observed with tdh negative Vibrio or non- Vibrio spp. The assay detected a single colony forming unit (CFU) per reaction of a pure culture template. This sensitivity was achieved when the same template amount per reaction was tested in the presence of 2.5 μl of a tdh negative oyster:APW enrichment (oyster homogenate enriched in alkaline peptone water overnight at 35 °C). This real time technique was used to test 131 oyster:APW enrichments from an environmental survey of Alabama oysters collected between March 1999 and September 2000. The results were compared to those previously obtained using a streak plate procedure for culture isolation from the oyster:APW enrichment combined with use of a non-radioactive DNA probe for detection of the tdh gene. Real time PCR detected tdh in 61 samples, whereas the streak plate/probe method detected tdh in 15 samples. Only 24 h was required for detection of pathogenic V. parahaemolyticus in oyster:APW enrichments by real time PCR, whereas the streak plate/probe method required 3 days and was more resource intensive. This study demonstrated that real time PCR is a rapid and reliable technique for detecting V. parahaemolyticus possessing the tdh gene in pure cultures and in oyster enrichments.