Purpose: To simplify the detection of norovirus (NoV) in food, a multiplex Q-RT-PCR was developed to identify simultaneously the 2 genogroups (I or II) of norovirus.
Methods: Primers and probes are in accordance with the specifications of the ISO/TS 15216. Optimizations have been performed to reach a sensitivity and an amplification efficiency comparable with the detection of only one norovirus genogroup per reaction. A robustness study was conducted. The detection method was validated on samples (50) previously found positive at various level of contamination for Nov GI or GII. Ready-to-use ceeramTools detection kits were produced and evaluated by 4 laboratories skilled for virus detection in food on various types of food samples.
Results: A limit of detection of 2.5 and 7 genome copies/reaction with a confidence level of 95% was reached respectively for NoV GI and GII. The robustness study demonstrated standard deviations below 0.8 for inter and intra-assays and inter manipulator variations. Using the complete workflow (sample prep, RNA extraction with NucliSens reagents, ceeramTools detection kits, GENE-UP cycler), all the previously positive food samples were found positive with the duplex detection even those with a level of contamination lower than 100 copies/25 g of food. Such results demonstrate the absence of significant difference between a simplex and a multiplex detection of NoV GI and GII.
Significance: This method allows a rapid detection of norovirus and identification of the genogroup in one reaction. The analytical costs can therefore be reduced. As more analyses can be performed for lower costs, more data one norovirus circulation and prevalence can be generated leading to a better viral food safety management. ceeramTools detection kits are also available for other targeted virus such as hepatitis A virus.