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Journal of Clinical Microbiology, September 2009, p. 2888-2894, Vol. 47, No. 9
0095-1137/09/$08.00+0     doi:10.1128/JCM.00792-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Development of a Multiplex PCR-Based Rapid Typing Method for Enterohemorrhagic Escherichia coli O157 Strains{triangledown} ,{dagger}

Tadasuke Ooka,1 Jun Terajima,2 Masahiro Kusumoto,3,6 Atsushi Iguchi,4 Ken Kurokawa,5 Yoshitoshi Ogura,1,4 Md Asadulghani,1 Keisuke Nakayama,1 Kazunori Murase,1 Makoto Ohnishi,2 Sunao Iyoda,2 Haruo Watanabe,2 and Tetsuya Hayashi1,4*

Division of Microbiology, Department of Infectious Diseases, Faculty of Medicine, University of Miyazaki, Miyazaki 889-1692, Japan,1 Department of Bacteriology, National Institute for Infectious Diseases, Tokyo 162-8640, Japan,2 Tsuruga Institute of Biotechnology, Toyobo Co. Ltd., Fukui 914-0047, Japan,3 Division of Bioenvironmental Science, Frontier Science Research Center, University of Miyazaki, Miyazaki 889-1692, Japan,4 Department of Biological Information, Tokyo Institute of Technology, Tokyo 152-8550, Japan,5 Safety Research Team, National Institute of Animal Health, Ibaraki 305-0856, Japan6

Received 20 April 2009/ Returned for modification 20 April 2009/ Accepted 15 July 2009

Enterohemorrhagic Escherichia coli O157 (EHEC O157) is a food-borne pathogen that has raised worldwide public health concern. The development of simple and rapid strain-typing methods is crucial for the rapid detection and surveillance of EHEC O157 outbreaks. In the present study, we developed a multiplex PCR-based strain-typing method for EHEC O157, which is based on the variability in genomic location of IS629 among EHEC O157 strains. This method is very simple, in that the procedures are completed within 2 h, the analysis can be performed without the need for special equipment or techniques (requiring only conventional PCR and agarose gel electrophoresis systems), the results can easily be transformed into digital data, and the genes for the major virulence markers of EHEC O157 (the stx1, stx2, and eae genes) can be detected simultaneously. Using this method, 201 EHEC O157 strains showing different XbaI digestion patterns in pulsed-field gel electrophoresis (PFGE) analysis were classified into 127 types, and outbreak-related strains showed identical or highly similar banding patterns. Although this method is less discriminatory than PFGE, it may be useful as a primary screening tool for EHEC O157 outbreaks.

* Corresponding author. Mailing address: Division of Bioenvironmental Science, Frontier Science Research Center, University of Miyazaki, Miyazaki 889-1692, Japan. Phone: 81-985-85-0871. Fax: 81-985-85-6475. E-mail: thayash{at}med.miyazaki-u.ac.jp

{triangledown} Published ahead of print on 29 July 2009.

{dagger} Supplemental material for this article may be found at http://jcm.asm.org/.

Journal of Clinical Microbiology, September 2009, p. 2888-2894, Vol. 47, No. 9
0095-1137/09/$08.00+0     doi:10.1128/JCM.00792-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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