title: Legionella in water samples: how can you interpret the results obtained by quantitative PCR?
abstract: Evaluation of the potential risk associated with Legionella has traditionally been determined from culture-based methods. Quantitative polymerase chain reaction (qPCR) is an alternative tool that offers rapid, sensitive and specific detection of Legionella in environmental water samples. In this study we compare the results obtained by conventional qPCR (iQ-Check™ Quanti Legionella spp.; Bio-Rad) and by culture method on artificial samples prepared in Page's saline by addiction of Legionella pneumophila serogroup 1 (ATCC 33152) and we analyse the selective quantification of viable Legionella cells by the qPCR-PMA method. The amount of Legionella DNA (GU) determined by qPCR was 28-fold higher than the load detected by culture (CFU). Applying the qPCR combined with PMA treatment we obtained a reduction of 98.5% of the qPCR signal from dead cells. We observed a dissimilarity in the ability of PMA to suppress the PCR signal in samples with different amounts of bacteria: the effective elimination of detection signals by PMA depended on the concentration of GU and increasing amounts of cells resulted in higher values of reduction. Using the results from this study we created an algorithm to facilitate the interpretation of viable cell level estimation with qPCR-PMA.
authors.affiliation: Department of Public Health and Pediatrics, University of Turin, P.zza Polonia, 94, 10126 Turin, Italy. Electronic address: savina.ditommaso@unito.it.
authors.forename: Savina
authors.initials: S
authors.lastname: Ditommaso
authors.affiliation: Department of Public Health and Pediatrics, University of Turin, P.zza Polonia, 94, 10126 Turin, Italy.
authors.forename: Elisa
authors.initials: E
authors.lastname: Ricciardi
authors.affiliation: Department of Public Health and Pediatrics, University of Turin, P.zza Polonia, 94, 10126 Turin, Italy.
authors.forename: Monica
authors.initials: M
authors.lastname: Giacomuzzi
authors.affiliation: Department of Public Health and Pediatrics, University of Turin, P.zza Polonia, 94, 10126 Turin, Italy.
authors.forename: Susan R
authors.initials: SR
authors.lastname: Arauco Rivera
authors.affiliation: Department of Public Health and Pediatrics, University of Turin, P.zza Polonia, 94, 10126 Turin, Italy.
authors.forename: Carla M
authors.initials: CM
authors.lastname: Zotti
mesh_terms: D000465:Algorithms; D001386:Azides / Q000494:pharmacology; D004269:DNA, Bacterial / Q000032:analysis; D016952:Legionella pneumophila / Q000187:drug effects / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D050296:Microbial Viability / Q000187:drug effects; D058889:Molecular Typing / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods*; D011419:Propidium / Q000031:analogs & derivatives / Q000494:pharmacology; D014871:Water Microbiology
chemical_list: D001386:Azides; D004269:DNA, Bacterial; C533957:propidium monoazide; D011419:Propidium
keywords: Enumeration; Environmental water; Legionella spp; PCR (polymerase chain reaction)