Anna Charlotte Schultz

Development and evaluation of novel one-step TaqMan realtime RT-PCR assays for the detection and direct genotyping of genogroup I and II noroviruses

BACKGROUNDnCurrent detection and genotyping methods of genogroup (G) I and II noroviruses (NoVs) consist of a 2-step approach including detection of viral RNA by TaqMan realtime RT-PCR (RT-qPCR) followed by conventional RT-PCR and sequencing of partial regions of ORF1 or ORF2.nnnOBJECTIVEnTo develop novel long-template one-step TaqMan assays (L-RT-qPCR) for the rapid detection and direct genotyping of GI and GII NoVs and to evaluate the sensitivity and specificity of the assays.nnnSTUDY DESIGNnGI and GII-specific broadly reactive L-RT-qPCR assays were developed by combining existing NoV primers and probes targeting the open reading frame (ORF)1-ORF2 junction as well as region C at the 5-ORF2. The assays were validated using GI and GII RNA transcripts and a coded panel of 75 stool samples containing NoV strains representing 9 GI genotypes and 12 GII genotypes, as well as sapoviruses, astroviruses, polioviruses, and rotaviruses. L-RT-qPCR products were typed by sequencing.nnnRESULTSnThe novel GI and GII L-RT-qPCR assays detected and typed all but one of the NoV positive panel samples. As few as 5-500 RNA copies could be accurately typed by sequencing of amplicons.nnnCONCLUSIONSnWe developed novel one-step TaqMan RT-qPCR assays for the sensitive detection and direct genotyping of GI and GII NoVs from clinical and environmental matrices.

Fourth Nordic Workshop on Food and Waterborne Viruses

Why are there no standardised methods for the detection of enteric viruses in food and water?This is a repeated question raised by the industry and food authority in particular in connection with t ...