Sensitive multiplex real-time reverse transcription-PCR assay for the detection of human and animal noroviruses in clinical and environmental samples

Abstract

In this study, we developed a triplex real-time reverse transcription-PCR (RT-PCR)-based method that detects and distinguishes between noroviruses belonging to genogroups I, II, and III and that targets the junction between the regions of open reading frame 1 (ORF1) and ORF2. This is the first assay to include all three genogroups and the first real-time RT-PCR-based method developed for the detection of bovine noroviruses. The assay was shown to be broadly reactive against a wide spectrum of norovirus genotypes, including GI/1 through GI/7, GII/1 through GII/8, GII/10, GII/12, and GII/17, in different matrices (including fecal specimens, treated and raw sewage, source water, and treated drinking water). The assay is highly sensitive, detecting low copy numbers of plasmids that carry the target sequence. A new bovine norovirus, Bo/NLV/Norsewood/2006/NZL, was identified by this assay and was further genetically characterized. The results implicate a broad range of possible applications, including clinical diagnostics, tracing of fecal contaminants, and due to its sensitivity and broad reactivity, environmental studies.

FIG. 1.

Neighbor-joining phylogenetic tree based on the partial ORF1 and ORF2 regions of bovine noroviruses. The newly identified strain (Bo/NLV/Norsewood/2006/NZL) is in boldface at the top of the tree.