Development of Conventional and Real-Time PCR Assays for Detection of Legionella DNA in Respiratory Specimens

Abstract

The development and validation of a PCR assay based on the use of new 16S ribosomal DNA (rDNA)-targeted primers to detect Legionella DNA in respiratory specimens are described. The assay was originally developed as conventional PCR followed by electrophoretic detection and was then adapted to Lightcycler format with SYBR Green I detection and melting curve analysis. The 73 Legionella pneumophila strains tested were amplified with both applications. In addition, 21 and 23 out of 27 other Legionella strains were found positive by conventional and real-time PCR assays, respectively, including the clinically important species L. micdadei, L. bozemaniae , and L. dumoffii . Two DNA purification methods were compared using artificially seeded clinical specimens: a standard organic extraction method and a commercial kit based on adsorption of DNA to silica particles. The detection limit of the assay varied from 2 CFU to >200,000 CFU per ml of clinical specimen, depending on the background sample (i.e., pooled sputa or BAL fluids) and the DNA purification method, the silica method achieving lower detection limits. Analysis of 77 clinical samples (66 bronchoalveolar lavage fluid and 11 sputum samples) by conventional PCR yielded results that were consistent with Legionella culture results. The melting curve analysis in the Lightcycler system readily detected the specific amplification products. However, run-to-run variations in the measured melting temperatures required normalization against the standard sample in each run. The results obtained with the clinical specimens were similar to those obtained with conventional PCR, but more samples are required to determine whether the system can be applied to routine screening of samples for the presence of Legionella DNA.