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Journal of Clinical Microbiology, November 2006, p. 3963-3968, Vol. 44, No. 11
0095-1137/06/$08.00+0     doi:10.1128/JCM.00717-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Comparison of a PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) Assay to Pulsed-Field Gel Electrophoresis To Determine the Effect of Repeated Subculture and Prolonged Storage on RFLP Patterns of Shiga Toxin-Producing Escherichia coli O157:H7{triangledown}

Kensuke Shima, Yuluo Wu, Norihiko Sugimoto, Masahiro Asakura, Kazuhiko Nishimura, and Shinji Yamasaki*

Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka, Japan

Received 5 April 2006/ Returned for modification 13 June 2006/ Accepted 14 August 2006

In this study, we compared a recently developed PCR-restriction fragment length polymorphism (PCR-RFLP) assay with pulsed-field gel electrophoresis (PFGE) using three different Shiga toxin-producing Escherichia coli (STEC) strains to understand whether repeated subculture in vitro and prolonged storage at room temperature affect the RFLP patterns of STEC. The PFGE profiles of the STEC strains changed by 1 to 8 fragments after repeated subculture and prolonged storage; one strain was no longer clonal after repeated subculture compared to the original isolate according to the Tenover criteria. In contrast, RFLP patterns obtained by PCR-RFLP were identical after repeated subculture and prolonged storage. These data clearly indicate that the PCR-RFLP assay which is based on the diversity of region V, a regulatory region of Stx-phage, was not affected by repeated subculture and prolonged storage and is a more practical and reliable method for molecular typing of STEC strains.

* Corresponding author. Mailing address: Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1, Gakuen-Cho, Naka-ku. Sakai, Osaka 599-8531, Japan. Phone and fax: 81 72 254 9515. E-mail: shinji{at}vet.osakafu-u.ac.jp.

{triangledown} Published ahead of print on 13 September 2006.

Journal of Clinical Microbiology, November 2006, p. 3963-3968, Vol. 44, No. 11
0095-1137/06/$08.00+0     doi:10.1128/JCM.00717-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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