Validation according to ISO/TS 12869:2012 of a molecular method for the isolation and quantification of Legionella spp. in water

none6noThe 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.openOmiccioli, Enrica; Schiavano, Giuditta Fiorella; Ceppetelli, Veronica; Amagliani, Giulia; Magnani, Mauro; Brandi, GiorgioOmiccioli, Enrica; Schiavano, GIUDITTA FIORELLA; Ceppetelli, Veronica; Amagliani, Giulia; Magnani, Mauro; Brandi, Giorgi

Validation and application of a quantitative real-time PCR assay to detect common wheat adulteration of durum wheat for pasta production

Pasta is the Italian product par excellence and it is now popular worldwide. Pasta of a superior quality is made with pure durum wheat. In Italy, addition of Triticum aestivum (common wheat) during manufacturing is not allowed and, without adequate labeling, its presence is considered an adulteration. PCRrelated techniques can be employed for the detection of common wheat contaminations. In this work, we demonstrated that a previously published method for the detection of T. aestivum, based on the gliadin gene, is inadequate. Moreover, a new molecular method, based on DNA extraction from semolina and real-time PCR determination of T. aestivum in Triticum spp., was validated. This multiplex real-time PCR, based on the dual-labeled probe strategy, guarantees target detection specificity and sensitivity in a short period of time. Moreover, the molecular analysis of common wheat contamination in commercial wheat and flours is described for the first time