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Applied and Environmental Microbiology, August 2003, p. 5023-5028, Vol. 69, No. 8
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.8.5023-5028.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Detection of Bacteria Carrying the stx2 Gene by In Situ Loop-Mediated Isothermal Amplification

Fumito Maruyama, Takehiko Kenzaka, Nobuyasu Yamaguchi, Katsuji Tani, and Masao Nasu*

Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan

Received 24 September 2002/ Accepted 27 May 2003

A new in situ DNA amplification technique for microscopic detection of bacteria carrying a specific gene is described. Loop-mediated isothermal amplification (LAMP) was used to detect stxA2 in Escherichia coli O157:H7 cells. The mild permeabilization conditions and low isothermal temperature used in the in situ LAMP method caused less cell damage than in situ PCR. It allowed use of fluorescent antibody labeling in the bacterial mixture after the DNA amplification for identification of E. coli O157:H7 cells with an stxA2 gene. Higher-contrast images were obtained with this method than with in situ PCR.

* Corresponding author. Mailing address: Graduate School of Pharmaceutical Sciences, Osaka University, 1-6, Yamada-oka, Suita, Osaka 565-0871, Japan. Phone: 81-6-6879-8170. Fax: 81-6-6879-8174. E-mail: nasu{at}phs.osaka-u.ac.jp.

Applied and Environmental Microbiology, August 2003, p. 5023-5028, Vol. 69, No. 8
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.8.5023-5028.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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