Use of a new seminested PCR combined with enzymatic digestion analysis for detection and differentiation of Legionella species and Legionella pneumophila in environmental water and sputum samples

A new seminested PCR combined with enzymatic digestion analysis method was developed for detection and differentiation of Legionella spp. and Legionella pneumophila in environmental water and sputum samples. The seminested PCR was a novel target for all Legionella testing; it detected 42 Legionella spp. and 12 other bacterial species without the cross-reactivity, and detected 1 fg of Legionella DNA in each PCR. The enzymatic digestion analysis was differentiated L. pneumophila from other Legionella spp.. The aim of this study was to investigate the usefulness of the new scheme for detection of L. pneumophila in environmental and sputum samples. A total of 24 water samples and 215 sputum samples were detected by the new scheme. Of 24 environmental water samples examined by this scheme and culture, 20 were found to be positive for Legionella spp.. Of these 20 samples, 85.0% (17 of 20) were positive by both PCR and culture, 10.0% (2 of 20) were positive by PCR alone, and 5.0% (1 of 20) was positive by culture alone. Among these 20 samples, 14 samples were positive for L. pneumophila by enzymatic digestion analysis, and they were verified by gene sequence analysis. Of the 215 sputum samples examined, PCR was found to be an essential diagnostic tool because only 6.25% (1 of 16) sputum samples were determined to be L. pneumophila by culture. The seminested PCR combined with enzymatic digestion analysis is an appropriate and useful method for Legionella surveillance. The combination of this scheme with culture method may be the best protocol for the detection of Legionella species in environmental water and sputum samples.