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Applied and Environmental Microbiology, October 2001, p. 4685-4693, Vol. 67, No. 10
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.10.4685-4693.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

TaqMan PCR for Detection of Vibrio cholerae O1, O139, Non-O1, and Non-O139 in Pure Cultures, Raw Oysters, and Synthetic Seawater†

W. J. Lyon*

Rapid Microbial Detection Facility, Department of Food Science, Louisiana State University Agricultural Center, Baton Rouge, Louisiana 70803

Received 7 May 2001/Accepted 25 June 2001

Vibrio cholerae is recognized as a leading human waterborne pathogen. Traditional diagnostic testing for Vibrio is not always reliable, because this bacterium can enter a viable but nonculturable state. Therefore, nucleic acid-based tests have emerged as a useful alternative to traditional enrichment testing. In this article, a TaqMan PCR assay is presented for quantitative detection of V. cholerae in pure cultures, oysters, and synthetic seawater. Primers and probe were designed from the nonclassical hemolysin (hlyA) sequence of V. cholerae strains. This probe was applied to DNA from 60 bacterial strains comprising 21 genera. The TaqMan PCR assay was positive for all of the strains of V. cholerae tested and negative for all other species of Vibrio tested. In addition, none of the other genera tested was amplified with the TaqMan primers and probe used in this study. The results of the TaqMan PCR with raw oysters and spiked with V. cholerae serotypes O1 and O139 were comparable to those of pure cultures. The sensitivity of the assay was in the range of 6 to 8 CFU g-1 and 10 CFU ml-1 in spiked raw oyster and synthetic seawater samples, respectively. The total assay could be completed in 3 h. Quantification of the Vibrio cells was linear over at least 6 log units. The TaqMan probe and primer set developed in this study can be used as a rapid screening tool for the presence of V. cholerae in oysters and seawater without prior isolation and characterization of the bacteria by traditional microbiological methods.

* Present address: Rapid Microbial Detection Facility, Baton Rouge, LA 70803. Phone: (225) 761-0919. E-mail: WandaLyon{at}msn.com.

dagger This is journal paper no. 120 of the Louisiana Agricultural Experiment Station, Baton Rouge, La. (project: S-263).

Applied and Environmental Microbiology, October 2001, p. 4685-4693, Vol. 67, No. 10
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.10.4685-4693.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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