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Applied and Environmental Microbiology, January 2009, p. 147-153, Vol. 75, No. 1
0099-2240/09/$08.00+0     doi:10.1128/AEM.00604-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Specific Detection of Viable Legionella Cells by Combined Use of Photoactivated Ethidium Monoazide and PCR/Real-Time PCR{triangledown}

Bin Chang,1 Kanji Sugiyama,2 Toshitsugu Taguri,3 Junko Amemura-Maekawa,1 Fumiaki Kura,1 and Haruo Watanabe1*

Department of Bacteriology, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku-ku, Tokyo 162-8640, Japan,1 Department of Microbiology, Shizuoka Institute of Environment and Hygiene, Kita'ando, Aoi-ku, Shizuoka-shi, Shizuoka 420-8637, Japan,2 Nagasaki Prefectural Institute for Environmental Research and Public Health, Ikeda 2-1306-11, Oomura-shi, Nagasaki 856-0026, Japan3

Received 13 March 2008/ Accepted 6 October 2008

Legionella organisms are prevalent in manmade water systems and cause legionellosis in humans. A rapid detection method for viable Legionella cells combining ethidium monoazide (EMA) and PCR/real-time PCR was assessed. EMA could specifically intercalate and cleave the genomic DNA of heat- and chlorine-treated dead Legionella cells. The EMA-PCR assay clearly showed an amplified fragment specific for Legionella DNA from viable cells, but it could not do so for DNA from dead cells. The number of EMA-treated dead Legionella cells estimated by real-time PCR exhibited a 104- to 105-fold decrease compared to the number of dead Legionella cells without EMA treatment. Conversely, no significant difference in the numbers of EMA-treated and untreated viable Legionella cells was detected by the real-time PCR assay. The combined assay was also confirmed to be useful for specific detection of culturable Legionella cells from water samples obtained from spas. Therefore, the combined use of EMA and PCR/real-time PCR detects viable Legionella cells rapidly and specifically and may be useful in environmental surveillance for Legionella.

* Corresponding author. Mailing address: Department of Bacteriology, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku-ku, Tokyo 162-8640, Japan. Phone: (81) 3-5285-1137. Fax: (81) 3-5285-1193. E-mail: haruwata{at}nih.go.jp

{triangledown} Published ahead of print on 31 October 2008.

Applied and Environmental Microbiology, January 2009, p. 147-153, Vol. 75, No. 1
0099-2240/09/$08.00+0     doi:10.1128/AEM.00604-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Delgado-Viscogliosi, P., Solignac, L., Delattre, J.-M. (2009). Viability PCR, a Culture-Independent Method for Rapid and Selective Quantification of Viable Legionella pneumophila Cells in Environmental Water Samples. Appl. Environ. Microbiol. 75: 3502-3512 [Abstract] [Full Text]  


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