# Norovirus Detection in Ready-To-Eat Salads by Propidium Monoazide Real Time RT-PCR Assay
## 1 Introduction  
Food and water-borne outbreaks reported in Europe in 2018 were related to bacterial agents (57.0% of all outbreaks), bacterial toxins (24.2%), and viruses (13.5% of all outbreaks). Among the causative agents, viruses are recognized as important emerging pathogens of food-borne disease [1]. The main food sources of enteric viruses were crustaceans, shellfish, mollusks, mixed food, buffet meals, water, fish and fishery products, and food of non-animal origin [2].  
During the last two decades, enteric viruses have frequently been detected in vegetables [3, 4]. In particular, leafy green vegetables and ready-to-eat (RTE) salads, consumed raw and typically with no further washing/decontamination procedures, are considered an important route of transmission of human enteric viruses [3, 5, 6]. Vegetables may become contaminated by enteric viruses during pre-harvest cultivation through the improper use of organic waste in agricultural land in the form of fertilizer, fecal pollution in irrigation waters, or through direct contamination by livestock, wild animals, and birds. In addition, improper food handling practices should be taken into consideration [4, 6, 7, 8].  
The current European legislation on microbiological criteria for foodstuffs, Commission Regulation (EC) No 2073/2005 [9], lays down food-safety criteria for relevant foodborne bacteria in pre-cut(ready-to-eat) fruit and vegetables, such as _Salmonella_ spp. and _Listeria monocytogenes_, and the process hygiene criteria, e.g., addressing _Escherichia coli_, applicable, respectively, to products placed on the market or to production processes [6, 8, 10]. Given the lack of any sufficiently-developed analytical methods and prevalence studies and quantitative data on viral occurrence in leafy green vegetables consumed raw, the legislation does not currently take into consideration the role of viruses as foodborne pathogens in this matrix [10].  
The current gold standard for detecting enteric viruses is reverse transcription real time PCR (real time RT-PCR) [11, 12]. Indeed, the horizontal method for determining the hepatitis A virus and norovirus in food uses real time RT-PCR for detection and quantification [13].  
Despite being a very sensitive technique, PCR has some limitations for food viral analysis in that it has trouble differentiating between infectious and inactivated viruses [14], consequently preventing an efficient risk assessment [15]. Viral infectivity is necessary to assess the cytopathic effects (CPE) in susceptible cell lines [16]. However, not all viruses, such as noroviruses, are able to grow in a cell line in vitro. Therefore, the use of nucleic acid intercalating dyes such as propidium monoazide (PMAxx), is a powerful problem-solving tool [14, 17]. Viability dyes are able to enter into damaged or destroyed capsids. Once it has penetrated, the dye intercalates covalently into RNA after exposure to strong visible light, thus interfering with DNA amplification. The rest of the genome, including that found in undamaged capsids, is unaffected by PMAxx and can be amplified with PCR [18, 19, 20].  
Given the considerable public health implications and very low infectious dose of human noroviruses (10-100 viral particles) [21], the aim of this work is to investigate the occurrence of genogroups GI and GII of norovirus (NoV) RNA in RTE slads following ISO 15216-2 (2017) and to provide an assessment of PMAxx real time RT-PCR assay feasibility in order to discriminate between native and inactivated noroviruses.