Development and Clinical Evaluation of an Internally Controlled, Single-Tube Multiplex Real-Time PCR Assay for Detection of Legionella pneumophila and Other Legionella Species

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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.