title: 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 microl of a tdh negative oyster:APW enrichment (oyster homogenate enriched in alkaline peptone water overnight at 35 degrees 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.
authors.affiliation: Gulf Coast Seafood Laboratory, U.S. Food and Drug Administration, Post Office Box 158, Dauphin Island, AL 36528-0158, USA. gblackstone@cfsan@fda.gov
authors.forename: George M
authors.initials: GM
authors.lastname: Blackstone
authors.forename: Jessica L
authors.initials: JL
authors.lastname: Nordstrom
authors.forename: Michael C L
authors.initials: MC
authors.lastname: Vickery
authors.forename: Michael D
authors.initials: MD
authors.lastname: Bowen
authors.forename: Richard F
authors.initials: RF
authors.lastname: Meyer
authors.forename: Angelo
authors.initials: A
authors.lastname: DePaola
mesh_terms: D000818:Animals; D001427:Bacterial Toxins; D015169:Colony Count, Microbial; D003470:Culture Media; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis; D005456:Fluorescent Dyes; D006460:Hemolysin Proteins / Q000235:genetics*; D010125:Ostreidae / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014736:Vibrio parahaemolyticus / Q000235:genetics / Q000302:isolation & purification* / Q000472:pathogenicity*; D014774:Virulence
chemical_list: D001427:Bacterial Toxins; D003470:Culture Media; D017931:DNA Primers; D004269:DNA, Bacterial; D005456:Fluorescent Dyes; D006460:Hemolysin Proteins; C068489:thermostable direct hemolysin