Qualitative detection of Legionella species in bronchoalveolar lavages and induced sputa by automated DNA extraction and real-time polymerase chain reaction.
Molecular assays for qualitative detection of Legionella spp. in clinical specimens were evaluated. DNA extraction was done either with a fully automated DNA extraction protocol on the MagNA Pure LC System or with manual DNA extraction. Amplification and detection were done by real-time polymerase chain reaction (PCR) on the LightCycler (LC) instrument. Oligonucleotides were derived from the 16S rRNA gene of Legionella spp. The assays included a specially designed DNA fragment as Legionella-specific internal control. For both molecular assays, the detection limit was determined to be 5 CFU per LC PCR run. Sixty-one clinical specimens were tested with the molecular assays. Results were compared to culture. Five samples were found to be positive with the molecular assays. Three of them were positive in culture. No inhibition was found throughout the whole study. In conclusion, the molecular assays described may lead to safe and early diagnosis of Legionnaires' disease. They proved to be suitable for the routine molecular diagnostics laboratory.
Raggam
R B
RB
Molecular Diagnostics Laboratory, Institute of Hygiene, KF-University Graz, Universitätsplatz 4, Austria.
Leitner
E
E
Mühlbauer
G
G
Berg
J
J
Stöcher
M
M
Grisold
A J
AJ
Marth
E
E
Kessler
H H
HH
D000328:Adult; D001331:Automation; D001992:Bronchoalveolar Lavage Fluid / Q000382:microbiology*; D002648:Child; D003199:Computer Systems; D004269:DNA, Bacterial / Q000032:analysis* / Q000302:isolation & purification; D005784:Gene Amplification; D006801:Humans; D016952:Legionella pneumophila / Q000235:genetics / Q000302:isolation & purification*; D007877:Legionnaires' Disease / Q000175:diagnosis*; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013183:Sputum / Q000382:microbiology*
D004269:DNA, Bacterial