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Applied and Environmental Microbiology, October 2005, p. 5702-5709, Vol. 71, No. 10
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.10.5702-5709.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Real-Time PCR Detection of Vibrio vulnificus in Oysters: Comparison of Oligonucleotide Primers and Probes Targeting vvhA

Gitika Panicker{dagger} and Asim K. Bej*

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

Received 25 June 2004/ Accepted 29 April 2005

We compared three sets of oligonucleotide primers and two probes designed for Vibrio vulnificus hemolysin A gene (vvhA) for TaqMan-based real-time PCR method enabling specific detection of Vibrio vulnificus in oysters. Two of three sets of primers with a probe were specific for the detection of all 81 V. vulnificus isolates by TaqMan PCR. The 25 nonvibrio and 12 other vibrio isolates tested were negative. However, the third set of primers, F-vvh1059 and R-vvh1159, with the P-vvh1109 probe, although positive for all V. vulnificus isolates, also exhibited positive cycle threshold (CT) values for other Vibrio spp. Optimization of the TaqMan PCR assay using F-vvh785/R-vvh990 or F-vvh731/R-vvh1113 primers and the P-vvh874 probe detected 1 pg of purified DNA and 103 V. vulnificus CFU/ml in pure cultures. The enriched oyster tissue homogenate did not exhibit detectable inhibition to the TaqMan PCR amplification of vvhA. Detection of 3 x 103 CFU V. vulnificus, resulting from a 5-h enrichment of an initial inoculum of 1 CFU/g of oyster tissue homogenate, was achieved with F-vvh785/R-vvh990 or F-vvh731/R-vvh1113 primers and P-vvh875 probe. The application of the TaqMan PCR using these primers and probe, exhibited detection of V. vulnificus on 5-h-enriched natural oysters harvested from the Gulf of Mexico. Selection of appropriate primers and a probe on vvhA for TaqMan-PCR-based detection of V. vulnificus in post-harvest-treated oysters would help avoid false-positive results, thus ensuring a steady supply of safe oysters to consumers and reducing V. vulnificus-related illnesses and deaths.

* Corresponding author. Mailing address: Department of Biology, University of Alabama at Birmingham, 1300 University Blvd., Campbell Hall, Rm. 102, Birmingham, AL 35294-1170. Phone: (205) 934-9857. Fax: (205) 975-6097. E-mail: abej{at}uab.edu.

{dagger} Present address: Centers for Disease Control and Prevention, HPV Section, MS-G41, 1600 Clifton Rd., Atlanta, GA 30333.

Applied and Environmental Microbiology, October 2005, p. 5702-5709, Vol. 71, No. 10
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.10.5702-5709.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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Copyright © 2005 by the American Society for Microbiology. All rights reserved.