Abstract:ObjectiveTo investigate the application of Legiolert enzyme-substrate method with mass spectrometry in direct quantitative detection and separation of Legionella pneumophila in environmental water samples. MethodsCollection of 14 public places in Nanjing from July 2021 to November 2021, including hotels and restaurants, leisure centers and other centralized air conditioning cooling water 18, shower water 30 and other total 48 samples. Legiolert enzyme-substrate method was used to detect Legionella pneumophila in water samples, and the isolated cultured pure colonies were identified for serological typing with mass spectrometry and fluorescence quantitative PCR test. The detection rate of Legionella pneumophila was compared with that by traditional isolated and cultured method. ResultsThe detection rates of Legionella pneumophila in 48 samples were 8.33% (4/48) by traditional method and 31.25% (15/48) by mass spectrometry and fluorescence quantitative PCR. There was a significantly difference in it between two detection methods (χ2＝5.37, P<0.05). ConclusionLegiolert enzyme-substrate method combined with mass spectrometry shows obviously higher positive detection rate of Legionella pneumophila in environmental water samples compared with the traditional isolation and cultivation．