title: Development of single-tube nested real-time PCR assays with long internally quenched probes for detection of norovirus genogroup II.
abstract: The high sequence variation of RNA viruses necessitates use of degenerate primers and probes or multiple primers and probes in molecular diagnostic assays. We showed previously that PCR amplification in two rounds, first with long target-specific primers and then with short generic primers, followed by detection using long probes, can tolerate sequence variation. Here we demonstrate that long primers and probes of up to 56 nucleotides can also be applied in real-time PCR for the detection of norovirus genogroup II with improved sensitivity. Probe design (method of incorporating quenchers, use of Zen internal quencher or traditional quenchers) greatly affects the sensitivity of the real-time PCR assays.
authors.affiliation: Section of Clinical Virology, Department of Medical Sciences, Uppsala University, Sweden.|Department of Biomedical Sciences and Veterinary Public Health, Swedish University of Agricultural Sciences, Uppsala, Sweden.
authors.forename: Hongyan
authors.initials: H
authors.lastname: Xia
authors.affiliation: Section of Clinical Virology, Department of Medical Sciences, Uppsala University, Sweden.|August Kirchenstein Institute of Microbiology and Virology, Riga Stradins University, Riga, Latvia.
authors.forename: Sabine
authors.initials: S
authors.lastname: Gravelsina
authors.affiliation: Section of Clinical Virology, Department of Medical Sciences, Uppsala University, Sweden.
authors.forename: Christina
authors.initials: C
authors.lastname: Öhrmalm
authors.affiliation: Department of Biomedical Sciences and Veterinary Public Health, Swedish University of Agricultural Sciences, Uppsala, Sweden.|Department of Risk and Benefit Assessment, National Food Administration, Uppsala, Sweden.
authors.forename: Jakob
authors.initials: J
authors.lastname: Ottoson
authors.affiliation: Section of Clinical Virology, Department of Medical Sciences, Uppsala University, Sweden.
authors.forename: Jonas
authors.initials: J
authors.lastname: Blomberg
mesh_terms: D017250:Caliciviridae Infections / Q000175:diagnosis / Q000821:virology*; D017931:DNA Primers / Q000235:genetics; D005838:Genotype; D006801:Humans; D029322:Norovirus / Q000235:genetics / Q000302:isolation & purification* / Q000472:pathogenicity; D057089:Pathology, Molecular; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*
chemical_list: D017931:DNA Primers; D012367:RNA, Viral
keywords: norovirus; real-time PCR; variation-tolerant capture multiplex assay