Quantitative RT-PCR for the enumeration of noroviruses (Norwalk-like viruses) in water and sewage

Abstract

Aims of investigation: (i) develop a quantitative RT-PCR for noroviruses and (ii) evaluate it on environmental samples. 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. 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. 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.