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Applied and Environmental Microbiology, April 2003, p. 2194-2200, Vol. 69, No. 4
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.4.2194-2200.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

PCR Detection of a Newly Emerged Pandemic Vibrio parahaemolyticus O3:K6 Pathogen in Pure Cultures and Seeded Waters from the Gulf of Mexico

Michael L. Myers,{dagger} Gitika Panicker, and Asim K. Bej*

Department of Biology, The University of Alabama at Birmingham, Birmingham, Alabama 35294-1170

Received 10 June 2002/ Accepted 6 January 2003

This study describes the optimization of PCR parameters and testing of a wide number of microbial species to establish a highly specific and sensitive PCR-based method of detection of a newly emerged pandemic Vibrio parahaemolyticus O3:K6 strain in pure cultures and seeded waters from the Gulf of Mexico (gulf water). The selected open reading frame 8 (ORF8) DNA-specific oligonucleotide primers tested were found to specifically amplify all 35 pathogenic V. parahaemolyticus O3:K6 pandemic isolates, whereas these primers were not found to detectably amplify two strains of V. parahaemolyticus O3:K6 that were isolated prior to the 1996 outbreaks, 122 non-O3:K6 strains of V. parahaemolyticus, 198 non-V. parahaemolyticus spp., or 16 non-Vibrio bacterial spp. The minimum level of detection by the PCR method was 1 pg of purified genomic DNA or 102 ORF8-positive V. parahaemolyticus O3:K6 cells in 100 ml of water. The effectiveness of this method for the detection of ORF8-positive isolates in environmental samples was tested in gulf water seeded with 10-fold serial dilutions of this pathogen. A detection level of 103 cells per 100 ml of gulf water was achieved. Also, the applicability of this methodology was tested by the detection of this pathogen in gulf water incubated at various temperatures for 28 days. This PCR approach can potentially be used to monitor with high specificity and well within the required range of sensitivity the occurrence and distribution of this newly emerged pathogenic V. parahaemolyticus O3:K6 strain in coastal, marine, and ship ballast waters. Early detection of V. parahaemolyticus O3:K6 will help increase seafood safety and decrease the risk of infectious outbreaks caused by this pathogen.

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

{dagger} Present address: U.S. Food and Drug Administration, Alameda, CA 94502-7070.

Applied and Environmental Microbiology, April 2003, p. 2194-2200, Vol. 69, No. 4
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.4.2194-2200.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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