Detection and Quantification of Norovirus in Raspberries Implicated in Disease Outbreaks

In recent years, raspberries have emerged as high risk foods for norovirus (NoV) outbreaks. However, the lack of robust and standardized methods has limited our understanding of the level of NoV contamination of raspberries correlated to public health risk. We present an optimized and quality controlled protocol for the viral RNA extraction using NucliSens and Plant RNA Isolation Aid and detection by RT-qPCR including bovine serum albumin of NoV in 25 g of raspberries. The protocol was applied on raspberries that had been linked epidemiologically to nine NoV outbreaks which had occurred in Denmark from 2009-2011. To minimize false negative results, mengovirus and murine norovirus were evaluated as sample process control viruses (SPCVs). Using the median RT-qPCR recovery for either SPCV±2 SD as quality criteria for successful extraction, 98% of samples were approved. The 50% limit of detection was 119 and 140 RT-qPCR units/25g for NoV GI or GII, respectively. Nineteen (37%) of 51 raspberry samples tested positive for GI and/or GII NoVs with the respective geometric mean values of 70 (range 22-330) or 29 (range 3-217) detectable genome copies/g of raspberries, when corrected using mengovirus recoveries. A 100% identical GI.6 sequence was detected in both raspberries and patient stool samples associated to one outbreak. In conclusion, this is the first report demonstrating quantified virus levels of contaminated raspberries linked to illness. These data can contribute to the development of quantitative risk assessments and improve our understanding of public health risk related to different levels of NoV contamination in raspberries.