AIMS: Aims of investigation: (i) develop a quantitative RT-PCR for noroviruses and (ii) evaluate it on environmental samples. METHODS AND RESULTS: Noroviruses in environmental water samples were concentrated by adsorption/elution/flocculation. Sewage was processed by clarification and protein flocculation. Norovirus-specific cDNA produced by primer-directed reverse transcription of extracted RNA was amplified by LightCycler and accumulation of product monitored by observation of fluorescence induced by the incorporation of SYBR Green. Absolute quantitation of product was achieved by construction of standard curves using quantitative standards produced by cloning a modified sequence of the 3'-region of the forward norovirus primer. Reaction specificity was confirmed by analysis of product melting curves. CONCLUSIONS: Sewage was found to contain up to 1.8 x 10(6) norovirus cDNA copies per 100 ml and effluent contained up to 1.7 x 10(6) copies per 10 l. Marine bathing water and recreational river waters also contained noroviruses. Sample inhibition was detected to varying degrees in most sample types. SIGNIFICANCE AND IMPACT OF THE STUDY: The study will enable quantitative comparisons be made of samples from different locations and treatment processes, and inform the debate on the revision of the EU Bathing Water Directive; it will have important implications for the analysis of samples derived from different aquatic matrices, and from foods.
AIMS: Aims of investigation: (i) develop a quantitative RT-PCR for noroviruses and (ii) evaluate it on environmental samples. METHODS AND RESULTS: Noroviruses in environmentalwater samples were concentrated by adsorption/elution/flocculation. Sewage was processed by clarification and protein flocculation. Norovirus-specific cDNA produced by primer-directed reverse transcription of extracted RNA was amplified by LightCycler and accumulation of product monitored by observation of fluorescence induced by the incorporation of SYBR Green. Absolute quantitation of product was achieved by construction of standard curves using quantitative standards produced by cloning a modified sequence of the 3'-region of the forward norovirus primer. Reaction specificity was confirmed by analysis of product melting curves. CONCLUSIONS: Sewage was found to contain up to 1.8 x 10(6) norovirus cDNA copies per 100 ml and effluent contained up to 1.7 x 10(6) copies per 10 l. Marine bathing water and recreational river waters also contained noroviruses. Sample inhibition was detected to varying degrees in most sample types. SIGNIFICANCE AND IMPACT OF THE STUDY: The study will enable quantitative comparisons be made of samples from different locations and treatment processes, and inform the debate on the revision of the EU Bathing Water Directive; it will have important implications for the analysis of samples derived from different aquatic matrices, and from foods.
Authors: Régis Pouillot; Jane M Van Doren; Jacquelina Woods; Daniel Plante; Mark Smith; Gregory Goblick; Christopher Roberts; Annie Locas; Walter Hajen; Jeffrey Stobo; John White; Jennifer Holtzman; Enrico Buenaventura; William Burkhardt; Angela Catford; Robyn Edwards; Angelo DePaola; Kevin R Calci Journal: Appl Environ Microbiol Date: 2015-05-01 Impact factor: 4.792
Authors: Elisabeth Mathijs; Ambroos Stals; Leen Baert; Nadine Botteldoorn; Sarah Denayer; Axel Mauroy; Alexandra Scipioni; Georges Daube; Katelijne Dierick; Lieve Herman; Els Van Coillie; Mieke Uyttendaele; Etienne Thiry Journal: Food Environ Virol Date: 2012-11-03 Impact factor: 2.778
Authors: Ambroos Stals; Elisabeth Mathijs; Leen Baert; Nadine Botteldoorn; Sarah Denayer; Axel Mauroy; Alexandra Scipioni; Georges Daube; Katelijne Dierick; Lieve Herman; Els Van Coillie; Etienne Thiry; Mieke Uyttendaele Journal: Food Environ Virol Date: 2012-11-04 Impact factor: 2.778