title: Specific detection of viable Legionella cells by combined use of photoactivated ethidium monoazide and PCR/real-time PCR.
abstract: 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 10(4)- to 10(5)-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.
authors.affiliation: Department of Bacteriology, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku-ku, Tokyo 162-8640, Japan.
authors.forename: Bin
authors.initials: B
authors.lastname: Chang
authors.forename: Kanji
authors.initials: K
authors.lastname: Sugiyama
authors.forename: Toshitsugu
authors.initials: T
authors.lastname: Taguri
authors.forename: Junko
authors.initials: J
authors.lastname: Amemura-Maekawa
authors.forename: Fumiaki
authors.initials: F
authors.lastname: Kura
authors.forename: Haruo
authors.initials: H
authors.lastname: Watanabe
mesh_terms: D001386:Azides / Q000378:metabolism*; D006801:Humans; D007875:Legionella / Q000235:genetics / Q000302:isolation & purification*; D008297:Male; D050296:Microbial Viability; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods*; D014871:Water Microbiology
chemical_list: D001386:Azides; C014535:8-azidoethidium