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Journal of Clinical Microbiology, November 2000, p. 4145-4151, Vol. 38, No. 11
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Rapid Method for Species-Specific Identification of Vibrio cholerae Using Primers Targeted to the Gene of Outer Membrane Protein OmpW

Bisweswar Nandi,1 Ranjan K. Nandy,1,2 Sarmishtha Mukhopadhyay,1 G. Balakrish Nair,2 Toshio Shimada,3 and Asoke C. Ghose1,*

Department of Microbiology, Bose Institute, Calcutta 700 054,1 and National Institute of Cholera and Enteric Diseases, Calcutta 700 010,2 India, and National Institute of Infectious Diseases, Tokyo 162, Japan3

Received 9 May 2000/Returned for modification 5 July 2000/Accepted 15 August 2000

The distribution of genes for an outer membrane protein (OmpW) and a regulatory protein (ToxR) in Vibrio cholerae and other organisms was studied using respective primers and probes. PCR amplification results showed that all (100%) of the 254 V. cholerae strains tested were positive for ompW and 229 (~98%) of 233 were positive for toxR. None of the 40 strains belonging to other Vibrio species produced amplicons with either ompW- or toxR-specific primers, while 80 bacterial strains from other genera tested were also found to be negative by the assay. These studies were extended with representative number of strains using ompW- and toxR-specific probes in DNA dot blot assay. While the V. cholerae strains reacted with ompW probe, only one (V. mimicus) out of 60 other bacterial strains tested showed weak recognition. In contrast, several strains belonging to other Vibrio species (e.g., V. mimicus, V. splendidus, V. alginolyticus, V. fluvialis, V. proteolyticus, V. aestuarianus, V. salmonicida, V. furnissii, and V. parahaemolyticus) showed weak to strong reactivity to the toxR probe. Restriction fragment length polymorphism analysis and nucleotide sequence data revealed that the ompW sequence is highly conserved among V. cholerae strains belonging to different biotypes and/or serogroups. All of these results suggest that the ompW gene can be targeted for the species-specific identification of V. cholerae strains. The scope of this study was further extended through the development of a one-step multiplex PCR assay for the simultaneous amplification of ompW and ctxA genes which should be of considerable value in the screening of both toxigenic and nontoxigenic V. cholerae strains of clinical as well as environmental origin.

* Corresponding author. Mailing address: Department of Microbiology, Bose Institute, P-1/12, CIT Scheme VII-M, Calcutta 700 054, India. Phone: 033-337-9416/9544/9219. Fax: 91-33-334-3886. E-mail: acghosh{at}boseinst.ernet.in.

Journal of Clinical Microbiology, November 2000, p. 4145-4151, Vol. 38, No. 11
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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