title: Development of a multiplex real-time PCR assay with an internal amplification control for the detection of total and pathogenic Vibrio parahaemolyticus bacteria in oysters.
abstract: Vibrio parahaemolyticus is an estuarine bacterium that is the leading cause of shellfish-associated cases of bacterial gastroenteritis in the United States. Our laboratory developed a real-time multiplex PCR assay for the simultaneous detection of the thermolabile hemolysin (tlh), thermostable direct hemolysin (tdh), and thermostable-related hemolysin (trh) genes of V. parahaemolyticus. The tlh gene is a species-specific marker, while the tdh and trh genes are pathogenicity markers. An internal amplification control (IAC) was incorporated to ensure PCR integrity and eliminate false-negative reporting. The assay was tested for specificity against >150 strains representing eight bacterial species. Only V. parahaemolyticus strains possessing the appropriate target genes generated a fluorescent signal, except for a late tdh signal generated by three strains of V. hollisae. The multiplex assay detected <10 CFU/reaction of pathogenic V. parahaemolyticus in the presence of >10(4) CFU/reaction of total V. parahaemolyticus bacteria. The real-time PCR assay was utilized with a most-probable-number format, and its results were compared to standard V. parahaemolyticus isolation methodology during an environmental survey of Alaskan oysters. The IAC was occasionally inhibited by the oyster matrix, and this usually corresponded to negative results for V. parahaemolyticus targets. V. parahaemolyticus tlh, tdh, and trh were detected in 44, 44, and 52% of the oyster samples, respectively. V. parahaemolyticus was isolated from 33% of the samples, and tdh(+) and trh(+) strains were isolated from 19 and 26%, respectively. These results demonstrate the utility of the real-time PCR assay in environmental surveys and its possible application to outbreak investigations for the detection of total and pathogenic V. parahaemolyticus.
authors.affiliation: Gulf Coast Seafood Laboratory, Division of Seafood Science and Technology, US Food and Drug Administration, P.O. Box 158, Dauphin Island, AL 36528-0158, USA. jessica.nordstrom@fda.hhs.gov
authors.forename: Jessica L
authors.initials: JL
authors.lastname: Nordstrom
authors.forename: Michael C L
authors.initials: MC
authors.lastname: Vickery
authors.forename: George M
authors.initials: GM
authors.lastname: Blackstone
authors.forename: Shelley L
authors.initials: SL
authors.lastname: Murray
authors.forename: Angelo
authors.initials: A
authors.lastname: DePaola
mesh_terms: D000818:Animals; D001427:Bacterial Toxins / Q000235:genetics; D015415:Biomarkers; D004269:DNA, Bacterial / Q000032:analysis; D005784:Gene Amplification; D006460:Hemolysin Proteins / Q000235:genetics*; D010125:Ostreidae / Q000382:microbiology*; D016133:Polymerase Chain Reaction; D014735:Vibrio Infections / Q000382:microbiology; D014736:Vibrio parahaemolyticus / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D001427:Bacterial Toxins; D015415:Biomarkers; D004269:DNA, Bacterial; D006460:Hemolysin Proteins; C068489:thermostable direct hemolysin