Development of a strand specific real-time RT-qPCR assay for the detection and quantitation of murine norovirus RNA

Murine norovirus (MNV), currently the only norovirus that efficiently replicates in cell culture, is often used as a model system to understand the molecular mechanisms of norovirus replication. MNV is a single stranded positive sense RNA virus of the Caliciviridae family. Replication of MNV involves the synthesis of both full length genomic and sub-genomic RNAs. The replication of these RNAs involves the synthesis of negative strand intermediates. To understand the molecular mechanism of RNA replication and the role of viral and host factors in virus replication, it is necessary to quantify accurately both positive and negative sense RNA molecules of the viral RNA during replication. Increasingly, strand specific reverse transcription-quantitative PCR (RT-qPCR) is becoming the method of choice for this kind of quantitation. Many strategies have been developed to avoid the false priming property of reverse transcriptase and to amplify specifically one strand in the presence of excess opposite strand. In this report, a SYBR based, real time RT-qPCR assay was developed to detect and quantify specifically the negative and the positive sense RNAs of MNV genomic RNA. This assay is based on using a tagged RT primer containing a non-viral sequence at the 5′ end of the viral strand specific sequence. This non-viral sequence is then used to amplify selectively the strand specific cDNA at the PCR stage. This assay can be used for a range of MNV strains including MNV-1 and 3, as these are now widely accepted for use in molecular studies. The specificity of this assay was determined by its ability to quantify one strand in the presence of up to 106 copies of competitor opposite sense RNA. Using this assay, the production of both strands of MNV-1 RNA was monitored during viral single step growth curve.

► Use of tagged primer to avoid amplification of false primed cDNA.
► Strand specific detection of norovirus genomic RNA.
► High sensitivity of the assay even in presence of excess opposite strand.