title: Development and clinical evaluation of an internally controlled, single-tube multiplex real-time PCR assay for detection of Legionella pneumophila and other Legionella species.
abstract: A multiplex real-time PCR assay for detection of Legionella pneumophila and Legionella spp. and including an internal control was designed. Legionella species, L. pneumophila, and the internal control were detected simultaneously by probes labeled with 6-carboxy-fluorescein, hexachlorofluorescein, and indodicarbocyanine, respectively. Therefore, no postamplification analysis was required in order to distinguish the targets. The sensitivity of both assays was 2.5 CFU/ml, and from analysis of 10 culture-positive and 74 culture-negative samples from patients investigated for legionellosis, 100% agreement was observed by both assays in comparison to culture. Four additional positives were found by the multiplex real-time PCR assay in the Legionella culture-negative samples.
authors.affiliation: Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Kate E
authors.initials: KE
authors.lastname: Templeton
authors.forename: Sitha A
authors.initials: SA
authors.lastname: Scheltinga
authors.forename: Peter
authors.initials: P
authors.lastname: Sillekens
authors.forename: Jantine W
authors.initials: JW
authors.lastname: Crielaard
authors.forename: Alje P
authors.initials: AP
authors.lastname: van Dam
authors.forename: Herman
authors.initials: H
authors.lastname: Goossens
authors.forename: Eric C J
authors.initials: EC
authors.lastname: Claas
mesh_terms: D006801:Humans; D007875:Legionella / Q000302:isolation & purification*; D016952:Legionella pneumophila / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity