title: Inhibition of Legionella pneumophila PCR in respiratory samples: a quantitative approach.
abstract: Impurities in complex biological samples that persist through nucleic acid preparation can inhibit PCR or reduce the sensitivity and efficiency of PCR amplification and thus affect reliable results in PCR diagnostics. To obtain information on the incidence of inhibition events and on the magnitude of the loss of sensitivity, respectively, we devised a relative inhibition assay and examined 100 samples from patients with respiratory tract infections. As a reference, samples were spiked with Legionella (L.) pneumophila. Detection was by standard nucleic acid purification and subsequent real-time PCR. By comparing the crossing points of the fluorescent curves to those of an L. pneumophila standard dilution series, we were able to quantify the respective degrees of inhibition into several categories. We found complete inhibition in 2% of the samples. 12% were not reliably detected. 65% of the tested samples showed moderate to strong inhibition, but were still reliably detected, whereas in 21% of the samples no inhibition was observed. Except for a significantly higher inhibition in tracheal aspirates than in BAL samples, the degree of inhibition did not correlate with the physical properties of the respective sample. The relative inhibition assay established an unexpectedly broad distribution of the inhibition-degrees in inflammatory respiratory materials.
authors.affiliation: Institute for Medical Microbiology and Hygiene, University of Regensburg, Franz-Josef-Strauss-Allee 11, Regensburg, Germany. monika.kern@klinik.uni-regensburg.de
authors.forename: Monika
authors.initials: M
authors.lastname: Kern
authors.forename: Stephan
authors.initials: S
authors.lastname: Böhm
authors.forename: Ludwig
authors.initials: L
authors.lastname: Deml
authors.forename: Hans
authors.initials: H
authors.lastname: Wolf
authors.forename: Udo
authors.initials: U
authors.lastname: Reischl
authors.forename: Hans Helmut
authors.initials: HH
authors.lastname: Niller
mesh_terms: D001992:Bronchoalveolar Lavage Fluid / Q000382:microbiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005188:False Negative Reactions; D006801:Humans; D016952:Legionella pneumophila / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012137:Respiratory System / Q000382:microbiology*; D012680:Sensitivity and Specificity
chemical_list: D004791:Enzyme Inhibitors