Legionella pneumophila serogroup 1 (sg1) accounts for the majority of infections in humans, but other
Legionella species are also associated with human disease. In this study, a new SYBR Green I-based
multiplex real-time PCR assay in a single reaction was developed to allow the rapid detection and differentiation
of Legionella species by targeting specific gene sequences. Candidate target genes were
selected, and primer sets were designed by referring to comparative genomic hybridization data of
Legionella species. The Legionella species-specific groES primer set successfully detected all 30 Legionella
strains tested. The xcpX and rfbA primers specifically detected L. pneumophila sg1e15 and L. pneumophila
sg1, respectively. In addition, this assay was validated by testing clinical samples and isolates. In
conclusion, this novel multiplex real-time PCR assay might be a useful diagnostic tool for the rapid
detection and differentiation of Legionella species in both clinical and epidemiological studies