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

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.

Assessment of hygienic conditions of recreational facility restrooms: an integrated approach.

INTRODUCTION: Microbiological quality of recreational environments included restrooms, is generally assessed by water and surface monitoring. In this study, an environmental monitoring, conducted in spring, of swimming pool restrooms of a recreation center located in the Marche region has been carried out. Seven water samples and seven surface swabs were collected. Moreover, six air samples have been included. The aim of this study was to evaluate if air microbiological monitoring, along with molecular detection in real-time PCR, could give additional useful information about the hygienic conditions of the facility. METHODS: Heterotrophic Plate Count (HPC) both at 22°C (psychrophilic) and 37°C (mesophilic) was determined by separate cultures in all samples. The presence of Legionella pneumophila and Pseudomonas aeruginosa was evaluated by both culture and real-time PCR. RESULTS: The analysis of shower water recorded a HPC load of mesophilic bacteria (37°C) more than 10-fold higher in men restroom, respect to women's one (> 100 vs < 10 CFU/ml), while in air samples was between < 100 and > 500. Concerning pathogen presence, both species Legionella pneumophila and Pseudomonas aeruginosa were detected only in men restroom, but in different sample types by using different methods (culture and real-time PCR). CONCLUSIONS: Air sampling may offer the advantage of giving more representative data about microbial presence in restrooms, including bacterial species transmitted through aerosol, like Legionella. Moreover, the concurrent use of molecular and microbiological detection in an integrated approach could offer the advantage of greater sensitivity.

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. PCR-related 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.