Development of a PCR Assay for the Detection of Legionella micdadei in the Environment

Background/Objectives: Legionella micdadei is a clinically significant species within the Legionella genus, requiring accurate detection methods, surveillance, and precise clinical diagnosis. Our objective was to develop a sensitive polymerase chain reaction (PCR) assay specific for L. micdadei to detect its presence in environmental specimens. Methods: We targeted the 23S–5S intergenic spacer region, which can differentiate Legionella spp. We tested the detection of L. micdadei with 20 strains and determined the limit of detection with 2 strains. We verified assay specificity with 17 strains of other Legionella spp., 62 strains of other bacterial and fungal genera, and three human DNA specimens. We evaluated intra- and inter-run precision. We tested 15 environmental specimens (water, swabs of water faucets, mulch, and soil) by PCR. Results: The PCR assay demonstrated 100% analytical specificity (no cross-reactivity with non-targeted species), 100% inclusivity (detection of all L. micdadei strains), and high precision, with a coefficient of variation ≤ 2% across replicates. The limit of detection was estimated at 5 genomic DNA copies per reaction. We detected L. micdadei in environmental specimens. Conclusions: This PCR assay enables accurate detection of L. micdadei and is not subject to competition with other Legionella spp., thereby addressing limitations of current broad-spectrum Legionella approaches. The evaluation supports its application in environmental detection for surveillance.