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Applied and Environmental Microbiology, September 2007, p. 5840-5847, Vol. 73, No. 18
0099-2240/07/$08.00+0     doi:10.1128/AEM.00460-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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{triangledown}

Jessica L. Nordstrom,1* Michael C. L. Vickery,1,{dagger} George M. Blackstone,1 Shelley L. Murray,2 and Angelo DePaola1

Gulf Coast Seafood Laboratory, Division of Seafood Science and Technology, U.S. Food and Drug Administration, Dauphin Island, Alabama 36528,1 Alaska Department of Environmental Conservation, Anchorage, Alaska 995012

Received 27 February 2007/ Accepted 12 July 2007

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 >104 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.

* Corresponding author. Mailing address: Division of Seafood Science and Technology, U.S. Food and Drug Administration, P.O. Box 158, Dauphin Island, AL 36528-0158. Phone: (251) 690-2341. Fax: (251) 694-4477. E-mail: jessica.nordstrom{at}fda.hhs.gov

{triangledown} Published ahead of print on 20 July 2007.

{dagger} Present address: Cepheid, 904 Caribbean Drive, Sunnyvale, CA 94089.

Applied and Environmental Microbiology, September 2007, p. 5840-5847, Vol. 73, No. 18
0099-2240/07/$08.00+0     doi:10.1128/AEM.00460-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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  • Zimmerman, A. M., DePaola, A., Bowers, J. C., Krantz, J. A., Nordstrom, J. L., Johnson, C. N., Grimes, D. J. (2007). Variability of Total and Pathogenic Vibrio parahaemolyticus Densities in Northern Gulf of Mexico Water and Oysters. Appl. Environ. Microbiol. 73: 7589-7596 [Abstract] [Full Text]  


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