# Norovirus Detection in Ready-To-Eat Salads by Propidium Monoazide Real Time RT-PCR Assay
## 4 Discussion
(2012) [33] found all ready-to-eat vegetable (100%) samples to be negative for Hepatitis A virus (HAV) while observing percentages of between 11.9% and 15.5% in RTE vegetables and red soft fruit, respectively, for NoV. In addition, [PERSON] et al. (2014) detected NoV in 13.6% of vegetable samples, lower than the results from our study. Therefore, the high occurrence of NoV observed in this study may have been biased by the limited sampling plan. Indeed, the samples tested for viral contamination came from only two RTE vegetable-processing plants. Therefore, the high presence of NoV makes it appropriate to investigate the water used on the two farms that supplied the samples of RTE salad subjected to analysis. Indeed, the high prevalence of viruses detected could potentially be an indicator of ineffective washing during the production process [34; 35]. Furthermore, it should be noted that these two vegetable-processing plants were chosen because they are the best-known brands on the Italian market and in order to sample different types of ready-to-eat salads. The findings in this study could also be due to virally-contaminated water, e.g., from an adjacent wastewater outflow, that was used in food production, processing, or preparation. Virus-contaminated water used for irrigation can be shifted from water to the surfaces of leafy green vegetables [36], causing the contamination of the vegetables. A further possible contamination factor for leafy green production enterprises, especially organic farms, is the use of manure, which can contain a variety of pathogens. Therefore, it is essential to successfully implement a Hazard Analisys and Critical Control Point (HACCP) plan, an effective way to ensure food safety from harvest to consumption. The identification of critical control points (CCPs), i.e., steps during which control can be applied, is crucial to avoiding or eliminating a food-safety hazard or decreasing it to an acceptable level. Further, the PMAxx real time RT-PCR results obtained in this study raise an important public health issue for the viral contamination of food in the vegetable-producing sector that should routinely adopt effective control tools for detecting viruses in foodstuffs. Specifically, in Commission Regulation (EC) 2073/05, the only microbiological criteria for establishing the acceptability of vegetables are conventional fecal indicators, whereas _Salmonella_ and _E. coli_ are untrustworthy for demonstrating the presence or absence of viruses at the end of the production chain (at retail) [28; 37]. This regulation does not take into consideration the risks derived from food contamination by enteric or emerging viral pathogens. Until recently, a major factor limiting the uptake of virus testing into regulatory food controls worldwide was the absence of any standardized and validated methods. As high sensitivity is needed, virus detection in foods is in general based on molecular methods such as reverse transcription polymerase chain reaction (RT-PCR) [28; 38]. Reverse transcription (RT)-PCR and, more recently, real time RT-PCR methods, reveal only the presence of viral nucleic acid in the samples investigated and cannot be used to discriminate the presence of infectious viral particles in the food matrices analyzed [6; 20]. From a sanitary point of view, the development of effective real time RT-PCR tools, useful not only for qualitative and quantitative detection but also for measuring virus infectivity, is an analytical priority. Therefore, the PMAxx real time RT-PCR assay described in this study, successfully applied to identify native and inactivated virus particles in RTE vegetables, assumes great analytical significance considering that, to the best our knowledge, this is the first study to detect the infectivity of NoV in naturally contaminated RTE salads.