title: Improved PCR assay for the species-specific identification and quantitation of Legionella pneumophila in water.
abstract: Legionellosis outbreak is a major global health care problem. However, current Legionella risk assessments may be compromised by uncertainties in Legionella detection methods, infectious dose, and strain infectivity. These limitations may place public health at significant risk, leading to significant monetary losses in health care. However, there are still unmet needs for its rapid identification and monitoring of legionellae in water systems. Therefore, in the present study, a primer set was designed based on a LysR-type transcriptional regulator (LTTR) family protein gene of Legionella pneumophila subsp. pneumophila str. Philadelphia 1 because it was found that this gene is structurally diverse among species through BLAST searches. The specificity of the primer set was evaluated using genomic DNA from 6 strains of L. pneumophila, 5 type strains of other related Legionella species, and other 29 reference pathogenic bacteria. The primer set used in the PCR assay amplified a 264-bp product for only targeted six strains of L. pneumophila. The assay was also able to detect at least 1.39 × 10(3) copies/μl of cloned amplified target DNA using purified DNA or 7.4 × 10(0) colony-forming unit per reaction when using calibrated cell suspension. In addition, the sensitivity and specificity of this assay were confirmed by successful detection of Legionella pneumophila in environmental water samples.
authors.affiliation: National Academy of Agricultural Science, Rural Development Administration, Jeonju, 560-500, Republic of Korea.|Department of Microbiology, Dankook University, Cheonan, 330-714, Republic of Korea.
authors.forename: Min Seok
authors.initials: MS
authors.lastname: Cho
authors.affiliation: Department of Microbiology, Dankook University, Cheonan, 330-714, Republic of Korea.
authors.forename: Tae-Young
authors.initials: TY
authors.lastname: Ahn
authors.affiliation: Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies, Wangsan, Yongin, Gyeonggi, 449-791, Republic of Korea.
authors.forename: Kiseong
authors.initials: K
authors.lastname: Joh
authors.affiliation: Department of Oral and Maxillofacial Surgery, Korea University Guro Hospital, Seoul, 152-703, Republic of Korea.
authors.forename: Eui Seok
authors.initials: ES
authors.lastname: Lee
authors.affiliation: National Academy of Agricultural Science, Rural Development Administration, Jeonju, 560-500, Republic of Korea. dspark@rda.go.kr.
authors.forename: Dong Suk
authors.initials: DS
authors.lastname: Park
mesh_terms: D058491:Bacterial Load / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D016952:Legionella pneumophila / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014871:Water Microbiology
chemical_list: D017931:DNA Primers
keywords: Detection; Legionella pneumophila; LysR transcriptional regulator; Real-time PCR