Use of bentonite-coated activated carbon for improving the sensitivity of RT-qPCR detection of norovirus from vegetables and fruits: The ISO 15216-1:2017 standard method extension.

Produce-related foodborne outbreaks are becoming increasingly prevalent worldwide. In plant tissues, various compounds, including polysaccharides, phenolic compounds, and chlorophyll, can inhibit RT-PCR detection of viruses. In this study, we developed a highly sensitive RT-qPCR in combination with the bentonite-coated activated carbon (BCAC) assay for detection of norovirus from fruits and vegetables, which could be completed within 7 h and was about 10-100 fold more sensitive than the standard procedures (ISO 15216-1:2017). The extraction efficiencies of three surrogate viruses (MS2, MNV-1, and TV) from five fresh produce (lettuce, cherry tomato, blueberry, strawberry, and spinach) were higher with BCAC treatment than those of control groups, ranging from 17.82% to 98.60%. The average detection limit of these viruses using the BCAC-RT-qPCR method was stable at an average of 102 PFU/g or GC/g. Finally, this BCAC-RT-qPCR method was applied for detection of human norovirus GII.4 spiked onto lettuce and cherry tomato. The viral extraction efficiencies were up to 53.43% and 95.56%, respectively, which is almost four and seven times better than those without BCAC. Therefore, the BCAC-RT-qPCR method can be used to detect low levels of foodborne viruses from produce.