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

Abstract

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 9 10 2 to 6.7 9 10 4 genome copies (GC) per millilitre; seawater samples contained from 7.6 9 10 1 to 2.4 9 10 3 per 10 l. As for clinical samples, the concentrations of NoVs per gram of stool varied, ranging from 6.1 9 10 3 to 1.4 9 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.

Detection of human norovirus GIV.1 in China: A case report

“…Similar to previous studies that reported high viral loads in patients with NoV gastroenteritis [31,32], a high viral load was detected in both of the patients with a GIV NoV infection. Based on these findings, it is suggested that a NoV GIV.1 infection can cause severe diarrhea in children under 5 years of age.…”

Analytical Methods for Food and Environmental Viruses

“…Even when used as a secondary step, ultracentrifugation has been shown to concentrate inhibitors (Casas et al 2007). Successful protocols have been developed using ultracentrifugation on highly purified preparations (La Rosa et al 2009). In addition, ultracentrifugation has the disadvantage of requiring expensive and highly specialized equipment.…”

A quantitative exposure model simulating human norovirus transmission during preparation of deli sandwiches

“…A first factor is the low presumed 50% infectious dose (ID 50 ) of 18 segregated infectious NoV particles (Teunis et al, 2008). Other factors include the shedding of high amounts of virus particles (up to 10 10 virus particles per gram feces) (Jeong et al, 2013;Lai et al, 2012;La Rosa et al, 2009;Atmar et al, 2008;Ozawa et al, 2007;Lee et al, 2007;Chan et al, 2006), a high environmental stability Wang et al, 2013;Baert et al, 2009a) and the frequent occurrence of asymptomatic infections (Jeong et al, 2013;Nicolay et al, 2011;Phillips et al, 2010). Regarding HuNoV post-harvest contamination of foods, a recent study found that in 42.5% of described HuNoV food borne outbreaks between 2000 and 2008 an infected food handler was responsible (Baert et al, 2009b).…”