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Journal of Clinical Microbiology, October 2003, p. 4676-4682, Vol. 41, No. 10
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.10.4676-4682.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Genotypic Analyses of Vibrio parahaemolyticus and Development of a Pandemic Group-Specific Multiplex PCR Assay

Masatoshi Okura,1 Ro Osawa,1* Atsushi Iguchi,1 Eiji Arakawa,2 Jun Terajima,2 and Haruo Watanabe2

Department of Bioscience, Graduate School of Science and Technology, Kobe University, Rokko-dai 1-1, Nada-ku, Kobe, 657-8501,1 Department of Bacteriology, National Institute of Infectious Diseases, Toyama, Shinjuku-ku, Tokyo, 162-8640, Japan2

Received 16 June 2003/ Accepted 10 July 2003

A total of 54 Vibrio parahaemolyticus strains including pandemic O3:K6 strains and newly emerged O4:K68, O1:K25, O1:K26, and O1:K untypeable strains (collectively referred to as the "pandemic group") were examined for their pulsed-field gel electrophoresis (PFGE) and arbitrarily primed PCR (AP-PCR) profiles and for the presence or absence of genetic marker DNA sequences, toxRS/new or orf8, that had been reported elsewhere to be specific for the pandemic group. Both PFGE and AP-PCR analyses indicated that all strains of the pandemic group formed a distinct genotypic cluster, suggesting that they originated from the same clone. In addition to the pandemic group, four O3:K6 strains that did not possess the thermostable direct hemolysin (tdh) gene also belonged to this cluster and possessed the toxRS/new sequence. However, three O3:K6 strains that clearly belonged to the pandemic group by PFGE and AP-PCR did not possess the orf8 sequence. The evidence suggests that neither the toxRS/new nor the orf8 sequence is a reliable gene marker for definite identification of the pandemic group. We therefore developed a novel multiplex PCR assay specific for the pandemic group. The assay successfully distinguished pandemic group strains from other V. parahaemolyticus strains by yielding two distinct PCR products for tdh (263 bp) and the toxRS/new sequence (651 bp).

* Corresponding author. Mailing address: Department of Bioscience, Graduate School of Science and Technology, Kobe University, Rokko-dai 1-1, Nada-ku, Kobe City 657-8501, Japan. Phone and fax: 81-78-803-5804. E-mail: osawa{at}ans.kobe-u.ac.jp.

Journal of Clinical Microbiology, October 2003, p. 4676-4682, Vol. 41, No. 10
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.10.4676-4682.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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