Significance of internal amplification control for the detection of human noroviruses from different food samples by real-time RT-PCR

Among human enteric viruses, the noroviruses (NoVs) are of a major epidemiological significance as a common cause of both epidemic and sporadic non-bacterial gastroenteritis in humans. According to the recent CDC data, NoVs caused most illnesses (58%) through consumed contaminated food especially soft fruits, vegetables and RTE foods. The most effective methods for virus detection in food samples are those based on nucleic acid amplification. However, two major obstacles that influence routine virus detection in foods include the low efficiency of concentration and nucleic acid extraction procedures and the presence of inhibitors from food matrix to the molecular reactions. Most false negatives are consequence of inefficient virus and/or nucleic acid extraction and of inhibition of the RT-PCR reaction. To verify whether amplification reactions have functioned correctly, implementation of internal amplification controls (IACs) is essential. For this purpose, previously constructed IACs for application with highly reactive degenerate primers (COG) for the detection of human NoVs genogroups GI and GII by real-time RT-PCR were tested. Twenty-five grams of raspberries, lettuce, cherry tomatoes, green onions and deli meat have been artificially inoculated with norovirus GI and GII positive stool samples. IACs were incorporated in all real-time RT-PCR assays, and simultaneous amplification of both target and RNA IAC was achieved after optimization without decreasing detection levels of virus. In this study it has been shown that IACs amplification was unaffected by the viral RNA extraction procedure from different food items, which could potentially be a source of diverse inhibitors