Journal of Microbiological Methods 2010-06-01

Rapid detection of Pseudomonas aeruginosa in mouse feces by colorimetric loop-mediated isothermal amplification

Motoki Goto, Kayo Shimada, Ayako Sato, Eri Takahashi, Takafumi Fukasawa, Tomoki Takahashi, Seii Ohka, Takahide Taniguchi, Eiichi Honda, Akio Nomoto, Atsuo Ogura, Teruo Kirikae, Ken-Ichi Hanaki, Motoki Goto, Kayo Shimada, Ayako Sato, Eri Takahashi, Takafumi Fukasawa, Tomoki Takahashi, Seii Ohka, Takahide Taniguchi, Eiichi Honda, Akio Nomoto, Atsuo Ogura, Teruo Kirikae, Ken-Ichi Hanaki

Index: J. Microbiol. Methods 81(3) , 247-52, (2010)

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Abstract

A colorimetric loop-mediated isothermal amplification (LAMP) assay with hydroxy naphthol blue was designed to amplify a region in the outer membrane lipoprotein (oprL) gene of Pseudomonas aeruginosa. The LAMP assay showed 100% specificity for the serogroup and other bacteria, and the sensitivity was 10-fold higher than that of the PCR assays. The LAMP assay could detect P. aeruginosa inoculated in mouse feces at 130 colony-forming units (CFU)/0.1g feces (3.25CFU/reaction). The assay was completed within 2h from DNA extraction. In a field trial, the LAMP assay revealed that none of the 27 samples was obtained from 2 specific pathogen-free (SPF) mouse facilities that were monitoring infection with P. aeruginosa; 1 out of 12 samples from an SPF mouse facility that was not monitoring infection with P. aeruginosa and 2 out of 7 samples from a conventional mouse facility were positive for P. aeruginosa. In contrast, P. aeruginosa was not detected in any of the samples by a conventional culture assay. Thus, this colorimetric LAMP assay is a simple and rapid method for P. aeruginosa detection.