Development of a strand specific SYBRGreen RT-PCR for a GIII.2 bovine norovirus

A strand specific SYBRGreen RT-PCR was developed for a bovine norovirus (GIII.2). HEK293 cells were transfected with a plasmid containing the complete virus genome and copy DNA was produced with viral RNA strand-specific primers that introduced nucleotide changes. Amplicons from the negative and positive viral RNA strands, and from potential transcripts made by sequence independent transcription, were separated by melting curve analysis. The RT-PCR showed high strand specificity and could be a useful tool to study virus replication in replicon and reverse genetic systems and in screening for low levels of virus replication in norovirus permissive cell lines. (C) 2013 Elsevier B.V. All rights reserved.