title: Validation according to ISO/TS 12869:2012 of a molecular method for the isolation and quantification of Legionella spp. in water.
abstract: The aim of the present work was to validate the performances of a new molecular method comprehensive of water sample filtration, DNA extraction and Real-Time PCR for the quantification of Legionella spp. in clear water samples, in accordance with the recent ISO Technical Specification 12869:2012. All criteria and requirements were verified considering inclusivity and exclusivity, check of the calibration function, limit of detection and limit of quantification, recovery calculation, robustness and uncertainty of the entire method. The performances were validated as all parameters resulted to be in compliance with values detailed by the above mentioned standard. The described method proved to be specific, sensitive, accurate and it has been fully validated according to ISO/TS 12869:2012. The possibility of using a validated molecular method will improve the reliability of the results making it a promising tool that should be used in addition to cultural analysis. Moreover, these findings make it particularly suitable for a relatively inexpensive screening of water samples, reducing the turnaround time and the workload.
authors.affiliation: Diatheva, Viale Piceno 137/F, Fano, PU, Italy. Electronic address: e.omiccioli@diatheva.com.
authors.forename: Enrica
authors.initials: E
authors.lastname: Omiccioli
authors.affiliation: Department of Biomolecular Sciences, Section of Hygiene, University of Urbino "Carlo Bo", Via santa Chiara 27, 61029, Urbino, PU, Italy.
authors.forename: Giuditta Fiorella
authors.initials: GF
authors.lastname: Schiavano
authors.affiliation: Diatheva, Viale Piceno 137/F, Fano, PU, Italy.
authors.forename: Veronica
authors.initials: V
authors.lastname: Ceppetelli
authors.affiliation: Department of Biomolecular Sciences, Section of Hygiene, University of Urbino "Carlo Bo", Via santa Chiara 27, 61029, Urbino, PU, Italy.
authors.forename: Giulia
authors.initials: G
authors.lastname: Amagliani
authors.affiliation: Department of Biomolecular Sciences, Section of Biochemistry and Molecular Biology, University of Urbino "Carlo Bo", Via Saffi 2, 61029, Urbino, PU, Italy.
authors.forename: Mauro
authors.initials: M
authors.lastname: Magnani
authors.affiliation: Department of Biomolecular Sciences, Section of Hygiene, University of Urbino "Carlo Bo", Via santa Chiara 27, 61029, Urbino, PU, Italy.
authors.forename: Giorgio
authors.initials: G
authors.lastname: Brandi
mesh_terms: D007875:Legionella / Q000235:genetics / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D014871:Water Microbiology
keywords: ISO/TS 12869; Legionella; PCR; Rapid method; Validation; Water quality