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.

Detection and Differentiation of Mycobacterium tuberculosis and Nontuberculous Mycobacterial Isolates by Real-Time PCR

“…The temperature at which the hybridization probes dissociated from their target sites was determined by melting curve analysis. This allowed for differentiation between species based on differences in the avidity of the hybridization probes for the complementary sequences in the amplified (24) Clinical isolates GC, BR, AP, LiPA, PS M. avium (17) Clinical isolates GC, BR, AP, PS M. intracellulare (8) Clinical isolates GC, BR, AP, PS M. marinum (22) Clinical isolates GC, BR, PS M. gordonae (17) Clinical isolates GC, BR, AP M. fortuitum (19) Clinical DNA. The melting curve for each specimen was analyzed manually to determine the melting temperature (T m ).…”

Evaluation of a Nested-PCR-Derived Sequence-Based Typing Method Applied Directly to Respiratory Samples from Patients with Legionnaires' Disease

“…However, some of these tests are too slow for clinical use and others are inadequately sensitive or specific (5). Real-time PCR performed with clinical samples has been positively evaluated in different laboratories for the diagnosis of Legionnaires' disease, suggesting that its addition to confirmed diagnostic methods such as culture and urinary antigen testing could significantly help in the early detection of additional patients with suspected legionellosis (6,16,18,21,29,30,32,35,37).…”

Development and Clinical Evaluation of an Internally Controlled, Single-Tube Multiplex Real-Time PCR Assay for Detection of Legionella pneumophila and Other Legionella Species

“…Therefore, clinically the identification of the actual Legionella species is required. Diagnostic PCR assays have principally targeted specific regions within 16S rRNA genes (rDNA) (3,9,12,21,23,26), 5S rDNA (9), or the macrophage inhibitor potentiator (mip) gene (1,9,15,16,17,20,22,26). Real-time PCR has added benefits to routine diagnosis, as it minimizes manual time for the PCR and negates the use of post-PCR analysis.…”