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Applied and Environmental Microbiology, March 2006, p. 2031-2042, Vol. 72, No. 3
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.3.2031-2042.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Detection of Vibrio parahaemolyticus in Shellfish by Use of Multiplexed Real-Time PCR with TaqMan Fluorescent Probes

Linda N. Ward1,2,{dagger} and Asim K. Bej1*

Department of Biology,1 Department of Justice Sciences, University of Alabama at Birmingham, Birmingham, Alabama2

Received 11 September 2005/ Accepted 19 December 2005

We developed a multiplexed real-time PCR assay using four sets of gene-specific oligonucleotide primers and four TaqMan probes labeled with four different fluorophores in a single reaction for detection of total and pathogenic Vibrio parahaemolyticus, including the pandemic O3:K6 serotype in oysters. V. parahaemolyticus has been associated with outbreaks of food-borne gastroenteritis caused by the consumption of raw or undercooked seafood and therefore is a concern to the seafood industry and consumers. We selected specific primers and probes targeting the thermostable direct hemolysin gene (tdh) and tdh-related hemolysin gene (trh) that have been reported to be associated with pathogenesis in this organism. In addition, we targeted open reading frame 8 of phage f237 (ORF8), which is associated with a newly emerged virulent pandemic serotype of V. parahameolyticus O3:K6. Total V. parahaemolyticus was targeted using the thermolabile hemolysin gene (tlh). The sensitivity of the combined four-locus multiplexed TaqMan PCR was found to be 200 pg of purified genomic DNA and 104 CFU per ml for pure cultures. Detection of an initial inoculum of 1 CFU V. parahaemolyticus per g of oyster tissue homogenate was possible after overnight enrichment, which resulted in a concentration of 3.3 x 109 CFU per ml. Use of this method with natural oysters resulted in 17/33 samples that were positive for tlh and 4/33 samples that were positive for tdh. This assay specifically and sensitively detected total and pathogenic V. parahaemolyticus and is expected to provide a rapid and reliable alternative to conventional detection methods by reducing the analysis time and obviating the need for multiple assays.

* Corresponding author. Mailing address: Department of Biology, University of Alabama at Birmingham, 1300 University Boulevard, Birmingham, AL 35294-1170. Phone: (205) 934-9857. Fax: (205) 975-6097. E-mail: abej{at}uab.edu.

{dagger} Present address: Identigene, Inc., 5615 Kirby Dr., Ste. 800, Houston, TX 77005.

Applied and Environmental Microbiology, March 2006, p. 2031-2042, Vol. 72, No. 3
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.3.2031-2042.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • 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]  
  • Nordstrom, J. L., Vickery, M. C. L., Blackstone, G. M., Murray, S. L., DePaola, A. (2007). 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. Appl. Environ. Microbiol. 73: 5840-5847 [Abstract] [Full Text]  
  • Nair, G. B., Ramamurthy, T., Bhattacharya, S. K., Dutta, B., Takeda, Y., Sack, D. A. (2007). Global Dissemination of Vibrio parahaemolyticus Serotype O3:K6 and Its Serovariants. Clin. Microbiol. Rev. 20: 39-48 [Abstract] [Full Text]  


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