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Applied and Environmental Microbiology, January 2004, p. 498-507, Vol. 70, No. 1
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.1.498-507.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Rapid Detection of Vibrio vulnificus in Shellfish and Gulf of Mexico Water by Real-Time PCR

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

Department of Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294

Received 24 April 2003/ Accepted 30 September 2003

In this paper we describe optimization of SYBR Green I-based real-time PCR parameters and testing of a large number of microbial species with vvh-specific oligonucleotide primers to establish a rapid, specific, and sensitive method for detection of Vibrio vulnificus in oyster tissue homogenate and Gulf of Mexico water (gulf water). Selected oligonucleotide primers for the vvh gene were tested for PCR amplification of a 205-bp DNA fragment with a melting temperature of approximately 87°C for 84 clinical and environmental strains of V. vulnificus. No amplification was observed with other vibrios or nonvibrio strains with these primers. The minimum level of detection by the real-time PCR method was 1 pg of purified genomic DNA or 102 V. vulnificus cells in 1 g of unenriched oyster tissue homogenate or 10 ml of gulf water. It was possible to improve the level of detection to one V. vulnificus cell in samples that were enriched for 5 h. The standard curves prepared from the real-time PCR cycle threshold values revealed that there was a strong correlation between the number of cells in unenriched samples and the number of cells in enriched samples. Detection of a single cell of V. vulnificus in 1 g of enriched oyster tissue homogenate is in compliance with the recent Interstate Shellfish Sanitation Conference guidelines. The entire detection method, including sample processing, enrichment, and real-time PCR amplification, was completed within 8 h, making it a rapid single-day assay. Rapid and sensitive detection of V. vulnificus would ensure a steady supply of postharvest treated oysters to consumers, which should help decrease the number of illnesses or 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, January 2004, p. 498-507, Vol. 70, No. 1
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.1.498-507.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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