Sensitive multiplex real-time reverse transcription-PCR assay for the detection of human and animal noroviruses in clinical and environmental samples.
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.
Wolf
Sandro
S
Communicable Disease Group, Institute of Environmental Science and Research Ltd., Kenepuru Science Centre, P.O. Box 50-348, Porirua, New Zealand.
Williamson
Wendy M
WM
Hewitt
Joanne
J
Rivera-Aban
Malet
M
Lin
Susan
S
Ball
Andrew
A
Scholes
Paula
P
Greening
Gail E
GE
D000818:Animals; D017250:Caliciviridae Infections / Q000821:virology*; D002417:Cattle; D005243:Feces / Q000821:virology; D005618:Fresh Water / Q000821:virology*; D005759:Gastroenteritis / Q000821:virology*; D006801:Humans; D008969:Molecular Sequence Data; D029322:Norovirus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D010802:Phylogeny; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D012722:Sewage / Q000821:virology*; D014881:Water Supply
D012722:Sewage