Abstract: Objective To establish a fluorescent quantitative polymerase chain reaction (real-time PCR) assay to detect the specific16S rRNA gene of Legionella in the sputum specimens patients with pulmonary Legionella infection. Methods 16S rRNA gene of Legionella was used to design primers and probes. The reaction system and reaction conditions were optimized. The specificity and sensitivity of the assay were evaluated by using Legionella pneumophila, non-Legionella pneumophila and other bacteria. The assay was used to detect 150 sputum specimens from patients with pulmonary Legionella infection, the positive ones were confirmed by gene sequencing of PCR products. Results The results showed that the results of 10 reference Legionella strains were all positive, but the results of other bacteria were all negative, and the assay's sensitivity was 10 cfu/ml. Among the 150 sputum specimens, 27 were positive for Legionella by fluorescent quantitative PCR (18.0%) and 22 were confirmed to be positive by gene sequencing (15.0%). The difference had no statistical significance (2=3.2, P0.05), indicating their good consistency and equivalence. Conclusion Real-time fluorescent quantitative PCR can be used to detect Legionella, it is specific and sensitive, and is sui Tablefor clinical investigation of Legionella infection and rapid detection.