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Gastroenteritis is a major public health problem worldwide, and human enteric viruses are the most common agents of foodborne gastroenteritis. Noroviruses (NoV), belonging to the Caliciviridae family, are pathogens that cause acute gastroenteritis. NoV are small nonenveloped viruses and have a positive-sense, single-stranded RNA genome, organized into three open reading frames (ORF). NoV are divided into five genogroups based on phylogenetic analysis of the capsid protein (Zheng et al. 2006). Genogroup I (GI), GII, and rarely GIV, are responsible for human outbreaks. NoV infection may induce vomiting, diarrhoea, mild fever, abdominal cramping and nausea in infected individuals. Moreover, noroviruses have recently been associated with multiple clinical outcomes other than gastroenteritis (Karst 2010). NoV are characterized by high environmental stability, and only few infectious viral particles are necessary to induce disease (Teunis et al. 2008). Transmission of these highly infectious viruses occurs mainly via the faecal–oral route, by ingestion of contaminated water and food, particularly shellfish, soft fruits and vegetables, but also through person-to-person contact and exposure to fomites (Beuchat 2006; Hewitt et al. 2007; Le Guyader et al. 2009; Lopman et al. 2012; Matthews et al. 2012; Zhou et al. 2012). NoV outbreaks occur most commonly in semi-closed communities such as restaurants, nursing homes, hospitals, schools, day care centres and cruise chips (Fankhauser et al. 2002).
Over the last few years, NoV diagnostics have improved, but there is currently no reliable method available for cultivation of NoV, and detection of this pathogen in foods relies primarily on molecular methods. Although enteric viruses have been detected in a range of samples, the main obstacles to routine detection of NoV in food and water samples are the low levels of virus contamination, variability in virus or nucleic acid extraction, the presence of substances that inhibit molecular detection and NoV genetic variability (Stals et al. 2012). Viral concentration methods in water include adsorption-elution methods using negatively or positively charged membrane filters, viral extraction and detection by real-time RT-PCR assays (Huguet et al. 2012).
Even if ISO/TS 15216-1 and 15216-2 methods have been recently published for a range of risk foods including soft fruits, bottled water and vegetables, they need to be further validated before publication as ISO/CEN standard methods. As recommended by ISO/TS 15216-1 and 15216-2, primers and probes for the individual quantification/detection of NoV GI and NoV GII should target the conserved ORF1/ORF2 junction of the genome. Another general requirement for viral diagnosis is the use of a process control to monitor the efficiency of the concentration of viral particles and the extraction of nucleic acid (Lees 2010). Although the TAG proposed the MC0 strain of Mengo virus (Costafreda et al. 2006; Le Guyader et al. 2009), there is as yet no consensus on the choice of process control (Lees 2010). The selected virus should exhibit similar morphological and physicochemical properties and environmental persistence to the target viruses, thus providing comparable extraction efficiency (Lees 2010). Ideally, the process control should be unlikely to naturally contaminate the tested food sample (Stals et al. 2012).
The first murine norovirus (MNV) was recently characterized and adapted to cell culture on murine macrophage-related cells (Karst et al. 2003; Wobus et al. 2004). MNV is morphologically and genetically similar to human noroviruses and shows considerable promise as a human norovirus surrogate (Wobus et al. 2006). On one hand, MNV-1 has been successfully tested as a process control when detecting NoV and HAV in some food samples (Stals et al. 2011a,b; Martin-Latil et al. 2012a) and HEV in bottled water (Martin-Latil et al. 2012b). On the other hand, the published method based on filtration of water and direct lysis of viruses on membrane has been found to be robust and may be useful and efficient for routine analyses of enteric viruses in bottled water (Martin-Latil et al. 2012ab; Perelle et al. 2009; Schultz et al. 2011).
With the aim of extending the use of a single process control for the detection of the main enteric viruses in food and water samples, the use of MNV-1 as a process control was evaluated for detecting NoV GI and NoV GII in bottled and tap water using filtration followed by a direct elution-based method. For this purpose, an one-step multiplex RT-qPCR was developed to detect NoV GI, NoV GII and MNV-1 simultaneously.
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- Materials and methods
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Noroviruses are considered to be the leading cause of foodborne and waterborne disease outbreaks and acute nonbacterial gastroenteritis worldwide, with transmission from food and water or from person to person via the faecal–oral route affecting adults and children all over the world (Atmar and Estes 2006; Koo et al. 2010). Indeed, NoV cause at least 95% of all nonbacterial gastroenteritis outbreaks and 50% of all gastroenteritis outbreaks (Karst 2010). Moreover, waterborne transmission is a significant route of exposure, as contaminated water also serves as a vehicle for outbreaks of food poisoning by foods such as vegetables and shellfish. Therefore, the availability of a sensitive, rapid and efficient virus concentration method for direct detection of viruses in drinkable water is an essential tool for the prevention of waterborne viral diseases.
The method used for virus extraction from water samples was previously described and evaluated through a collaborative trial for the detection of HAV, NoV and FCV in bottled water (Schultz et al. 2011). This method has been preferred to the ISO/TS 15216 method, as the authors clearly showed its efficiency, reproducibility and robustness across laboratories. This method is a two-step procedure: viruses are concentrated using positive-charged membranes, and the adsorbed viruses are directly lysed on membrane followed by application of automatic RNA extraction equipment. Multiple pathogen detection represents an option to decrease reagent costs and speed up the detection of several pathogens in a single reaction. Therefore, in the current study, a quantitative one-step sensitive multiplex RT-qPCR assay was first characterized for the simultaneous detection of NoV (GI and GII) and MNV-1 chosen as a process control, to be further used to detect and quantify NoV and MNV-1 in water samples.
We used primers and hydrolysis probes recommended for NoV detection by the European Committee for Standardization. These primer sets were recently shown to be among the most sensitive primer sets for NoV GI and NoV GII detection (Tong et al. 2011). With the one-step singleplex and multiplex RT-qPCR developed in this study, we also showed a higher sensitivity (about 1 log10) for detecting NoV GII than for detecting NoV GI. The higher sensitivity for NoV GII detection could be explained by the molecular model by itself. However, Stals et al. (2009) have developed a quantitative two-step multiplex real-time reverse transcriptase PCR assay to detect NoV and MNV-1, which was as sensitive for NoV GI as for NoV GII (LOD of 10 genome copies per assay). This higher sensitivity for NoV GI detection may be explained by the fact that a two-step RT-qPCR method could be more efficient and sensitive than an one-step method. However, we preferred an one-step protocol to minimize handling and therefore reduce chances of pipetting errors and cross-contamination. Moreover, unlike the results obtained by Stals et al. (2009), there was no significant competitive effect between the NoV GI and NoV GII reactions. In this study, the multiplex assay was found reliable for detecting NoV GI and NoV GII within the same sample based on the range of Ct shifts and parameters of regression lines.
The higher sensitivity for detecting NoV GII than for NoV GI has been also found to detect NoV regardless of the type of water. We also noticed an influence of the inoculum level on the recovery efficiency of NoV GI and NoV GII as previously shown by Stals et al. (2011a,b) on frozen raspberries and by Fumian et al. (2009) on lettuce. In addition, the variability between experiments was observed for inoculation levels equal and/or below the LOD where recovery rates were the highest. This interassay variability was significant for NoV GII detection and has also been observed for the detection of various micro-organisms in food (Le Guyader et al. 2009; Postollec et al. 2011; Martin-Latil et al. 2012a). We found significant improvement in NoV GI extraction yields calculated with diluted RNA samples (factor 1·5), which was not observed in the case of NoV GII detection. Direct lysis of viral particles on filtration membranes should limit loss of viral particles, but inhibitors may be also concentrated (Gregory et al. 2006; da Silva et al. 2007), and their effects on PCR amplification may vary according to viral targets. Nevertheless, the LOD of the method for detecting NoV was not improved after sample dilution. Therefore, at very low levels of contamination, dilution itself probably brings the sample below levels of detection, with a very limited impact of inhibitory substances for the detection of NoV in water.
Unlike the norovirus target, the type of water had a significant effect on the recovery rates of MNV-1 used as a control process, as the ratio of marginal means was 2·4 times higher in bottled water than in tap water. The influence of water type has already been observed in the detection of enteric viruses such as HAV, HEV and poliovirus (Blaise-Boisseau et al. 2010; Huguet et al. 2012; Martin-Latil et al. 2012b). However, recovery rates obtained for NoV extraction were quite similar regardless of the water type tested, in agreement with Huguet et al. (2012), showing that tap water quality did not affect the analytical performance of molecular methods for detecting NoV.
As shown in our previous study, the inhibition of MNV-1 amplification was slightly more pronounced in tap water than in bottled water (Martin-Latil et al. 2012b). Overall, it would appear that NoV are detected consistently with the same range of recovery rates of MNV-1, showing that MNV-1 is a robust option for routine sample processes in tap and bottled water analysis and should be further tested for other type of water.
In conclusion, the method described provides a valuable tool for the monitoring of potential public health risks associated with NoV contamination in drinkable water. It could be further interesting to determine the recovery efficiencies of NoV obtained using the described method throughout the different types of environmental water. Given the increasing evidence for NoV involvement in food outbreaks, the one-step triplex we used in this study would be a very useful tool to investigate NoV contamination in other food products.