DEVELOPMENT OF A NEW SEMINESTED PCR METHOD FOR DETECTION OF LEGIONELLA SPECIES AND ITS APPLICATION TO SURVEILLANCE OF LEGIONELLAE IN-HOSPITAL COOLING-TOWER WATER

The presence of PCR inhibitors in water samples is well known and cont ributes to the fact that a practical PCR assay has not been developed for legionella surveillance. In this study, we devised a new semineste d PCR assay for detection of Legionella spp. in water samples as a mea ns of overriding the PCR inhibitors without loss of sensitivity. The s eminested PCR assay utilized primers to amplify the 16S rRNA gene (LEG primers) of 39 Legionella spp. The assay was specific to legionellae, and the sensitivity was 1 fg of extracted Legionella DNA in laborator y examination, To evaluate the feasibility and sensitivity of the PCR assay in identifying the presence of legionellae, it was used to surve y Legionella contamination in the water of 49 cooling towers of 32 hos pitals. A commercially available EnviroAmp Legionella kit and a cultur e method were also used in the survey for comparison with the seminest ed PCR assay. The detection rates of legionellae in the samples were 9 1.8% (45 of 49) by the PCR assay and 79.5% (39 of 49) by the culture m ethod. The EnviroAmp kit revealed that 30.6% of the water samples (15 of 49) contained inhibitors of the PCR amplification. However, the sem inested PCR assay could produce the Legionella-specific DNA bands in 1 4 of the 15 samples. Although 8 of the 14 samples were positive in the first-step PCR, 6 of the 14 samples became positive in the second-ste p PCR. These results suggest that the effect of PCR inhibitors in samp les, if any, can be reduced because of the dilution of the sample in t he second-step PCR and that sensitivity of detection can be increased by the second-step PCR. Thus, the seminested PCR assay with LEG primer s to amplify the 16S rRNA gene of 39 Legionella spp. was a practical a nd sensitive method to detect Legionella spp. in water samples.