# Norovirus Detection in Ready-To-Eat Salads by Propidium Monoazide Real Time RT-PCR Assay
## 4 Discussion  
Noroviruses are frequently being identified as common causes of acute gastroenteritis (AGE), and their true burden is largely underestimated [25]. Noroviruses may contaminate food at any stage in the food supply chain, and transmission to humans can take place by ingestion of food contaminated either during the production process (during either primary production or further processing), or else by infected food handlers. Food can act as a vehicle for transmitting viruses to people. Enteric viruses will not propagate outside a host nor in foods, but may remain for a long time as infectious particles in food, and can live on in an infectious state until the foods are consumed [26]. Although food-borne NoV outbreaks have largely been linked to shellfish consumption in Europe [27; 28], other matrices such as frozen red fruits have recently been associated with such infections [29; 30]. Moreover, viruses have been implicated in cases of food-borne illness linked with contaminated leafy green vegetables and related ready-to-eat products [6; 31; 32]. \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Viruses** & **Treatment** & **Ct Value** & **Reduction** \\ \hline \multirow{4}{*}{**NoV GI**} & NT & 28.66 \(\pm\) 0.23 & - \\  & PMAxx [50 \(\upmu\)M] & 33.79 \(\pm\) 0.19 & \(\mathbf{-5.12\pm 0.37\) *} \\  & 95 \({}^{\circ}\)C & 32.79 \(\pm\) 0.19 & \(\mathbf{-3.92\pm 0.18}\) \\  & 95 \({}^{\circ}\)C + PMAxx [50 \(\upmu\)M] & 36.35 \(\pm\) 0.78 & \(\mathbf{-7.69\pm 0.96\) *} \\ \hline \multirow{4}{*}{**NoV GII**} & NT & 28.59 \(\pm\) 0.31 & - \\  & PMAxx [50 \(\upmu\)M] & 32.70 \(\pm\) 0.18 & \(\mathbf{-4.10\pm 0.27\) *} \\ \cline{1-1}  & 95 \({}^{\circ}\)C & 33.41 \(\pm\) 0.32 & \(\mathbf{-4.82\pm 0.52}\) \\ \cline{1-1}  & 95 \({}^{\circ}\)C + PMAxx [50 \(\upmu\)M] & 36.70 \(\pm\) 0.25 & \(\mathbf{-8.06\pm 0.27\) *} \\ \hline \hline \end{tabular} Legend: NT = not treated; PMAxx = propidium monoazide. * \(p\) value \(<\) 0.05.  
\end{table} 
Table 1: Results of real time RT-PCR for norovirus (NoV) genotypes I and II (GI and GII) detection after different treatment. Table explanation: The significant values for method validation are as follows: The first value of reduction rate (\(-\)5.12) refers to the NoV GI treated only with PMAxx compared with the non-treated NoV GI; the third value of reduction rate (\(-\)7.69) refers to the NoV GI treated with PMAxx and high temperature compared with the non-treated NoV GI. In the same way, it is possible observe a reduction rate of \(-\)4.10 between NoV GII treated with only PMAxx and the non-treated NoV GII and a reduction rate of \(-\)8.06 between NoV GII treated with PMAxx and high temperature and the non-treated NoV GII. \begin{table}
\begin{tabular}{c c c} \hline \hline \multirow{2}{*}{**Samples**} & \multicolumn{2}{c}{**NoV GII Positive**} \\ \cline{2-3}  & **Pre-Treatment** & **Post-Treatment** \\ \hline
**Mixed salad** & 17/21 (80.9\%) & 6/21 (28.6\%) \\
**Carrot** & 8/9 (88.8\%) & 2/9 (22.2\%) \\
**Valerian** & 27/33 (81.8\%) & 9/33 (27.3\%) \\
**Rocket** & 5/9 (55.6\%) & 0/9 \\
**Spinach** & 15/15 (100\%) & 6/15 (40\%) \\
**Iceberg Lettuce** & 15/21 (71.4\%) & 9/21 (42.9\%) \\
**Romaine Lettuce** & 0/6 & 0/6 \\
**Curly Leaf Endive** & 8/9 (88.9\%) & 3/9 (33.3\%) \\
**Chicory** & 6/9 (66.7\%) & 3/9 (33.3\%) \\
**Trevisano Chicory** & 0/3 & 0/3 \\
**Total Samples** & 101/135 (74.8\%) & 38/135 (28.1\%) \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Results of NoVs GII RNA detection in ready-to-eat (RTE) salad pre- and post-treatment with PMAxx [50 \(\upmu\)M]. In this study, the high prevalence of NoV in RTE salads, observed using the standard method for NoV in foodstuffs developed by the European Committee of Standardization (ISO/TS 15216-2:2017), may explain the data from the European Food Safety Authority reporting a rise in the number of NoV outbreaks due to consumption of vegetables in Europe [2]. However, the data on the occurrence of NoV in the present study are higher than those reported by other authors. Specifically, [PERSON] et al.