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Appl Environ Microbiol, February 1998, p. 681-687, Vol. 64, No. 2
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Cloning and Nucleotide Sequence of the gyrB Gene of Vibrio parahaemolyticus and Its Application in Detection of This Pathogen in Shrimp

Kasthuri Venkateswaran,1,* Nobuhiko Dohmoto,1 and Shigeaki Harayama2

Nippon Suisan Kaisha, Ltd., Central Research Laboratory, Hachioji City, Tokyo 192,1 and Marine Biotechnology Institute, Kamaishi Laboratories, Kamaishi, Iwate 026,2 Japan

Received 3 October 1997/Accepted 1 December 1997

Because biochemical testing and 16S rRNA sequence analysis have proven inadequate for the differentiation of Vibrio parahaemolyticus from closely related species, we employed the gyrase B gene (gyrB) as a molecular diagnostic probe. The gyrB genes of V. parahaemolyticus and closely related Vibrio alginolyticus were cloned and sequenced. Oligonucleotide PCR primers were designed for the amplification of a 285-bp fragment from within gyrB specific for V. parahaemolyticus. These primers recognized 117 of 117 reference and wild-type V. parahaemolyticus strains, whereas amplification did not occur when 90 strains of 37 other Vibrio species or 60 strains representing 34 different nonvibrio species were tested. In 100-µl PCR mixtures, the lower detection limits were 5 CFU for live cells and 4 pg for purified DNA. The possible application of gyrB primers for the routine identification of V. parahaemolyticus in food was examined. We developed and tested a procedure for the specific detection of the target organism in shrimp consisting of an 18-h preenrichment followed by PCR amplification of the 285-bp V. parahaemolyticus-specific fragment. This method enabled us to detect an initial inoculum of 1.5 CFU of V. parahaemolyticus cells per g of shrimp homogenate. By this approach, we were able to detect V. parahaemolyticus in all of 27 shrimp samples artificially inoculated with this bacterium. We present here a rapid, reliable, and sensitive protocol for the detection of V. parahaemolyticus in shrimp.

* Corresponding author. Present address: Jet Propulsion Laboratory, 4800 Oak Grove Dr., Pasadena, CA 91109. Phone: (818) 354-9219. Fax: (818) 393-6546. E-mail: kjvenkat{at}csd.uwm.edu.




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