Quantification of Norovirus Genogroups I and II in Environmental and Clinical Samples Using TaqMan Real-Time RT-PCR

Noroviruses (NoVs) are a major cause of acute non-bacterial gastroenteritis in all age groups. They efficiently circulate in both clinical and environmental contexts. In this study, real-time RT-PCR methods based on TaqMan probe technology were used to enumerate human noroviruses (genogroups I and II) in clinical samples, and in estuarine, seawater and sewage water samples, with the aim of obtaining quantitative information on the level of viral contamination. This was achieved through a quantitative analysis of the highly conserved region between ORF1 and ORF2, using genogroup-specific assays. RNA standards used to construct calibration curves for the two genogroups were generated by in vitro transcription from recombinant pCR4TOPO vectors containing a partial sequence coding for RdRp polymerase. Sewages were found to contain from 6.8 x 10(2) to 6.7 x 10(4) genome copies (GC) per millilitre; seawater samples contained from 7.6 x 10(1) to 2.4 x 10(3) per 10 l. As for clinical samples, the concentrations of NoVs per gram of stool varied, ranging from 6.1 x 10(3) to 1.4 x 10(8). Real-time PCR is an easy to use, sensitive and specific tool able to generate quantitative data, and could prove useful in both environmental and clinical settings.