title: Development and evaluation of novel one-step TaqMan realtime RT-PCR assays for the detection and direct genotyping of genogroup I and II noroviruses.
abstract: BACKGROUND
  Current detection and genotyping methods of genogroup (G) I and II noroviruses (NoVs) consist of a 2-step approach including detection of viral RNA by TaqMan realtime RT-PCR (RT-qPCR) followed by conventional RT-PCR and sequencing of partial regions of ORF1 or ORF2.
  
  
  OBJECTIVE
  To develop novel long-template one-step TaqMan assays (L-RT-qPCR) for the rapid detection and direct genotyping of GI and GII NoVs and to evaluate the sensitivity and specificity of the assays.
  
  
  METHODS
  GI and GII-specific broadly reactive L-RT-qPCR assays were developed by combining existing NoV primers and probes targeting the open reading frame (ORF)1-ORF2 junction as well as region C at the 5'-ORF2. The assays were validated using GI and GII RNA transcripts and a coded panel of 75 stool samples containing NoV strains representing 9 GI genotypes and 12 GII genotypes, as well as sapoviruses, astroviruses, polioviruses, and rotaviruses. L-RT-qPCR products were typed by sequencing.
  
  
  RESULTS
  The novel GI and GII L-RT-qPCR assays detected and typed all but one of the NoV positive panel samples. As few as 5-500 RNA copies could be accurately typed by sequencing of amplicons.
  
  
  CONCLUSIONS
  We developed novel one-step TaqMan RT-qPCR assays for the sensitive detection and direct genotyping of GI and GII NoVs from clinical and environmental matrices.
authors.affiliation: The National Food Institute, Technical University of Denmark, Mørkhøj Bygade 19, DK-2860 Søborg, Denmark. acsc@food.dtu.dk
authors.forename: Anna Charlotte
authors.initials: AC
authors.lastname: Schultz
authors.forename: Everardo
authors.initials: E
authors.lastname: Vega
authors.forename: Anders
authors.initials: A
authors.lastname: Dalsgaard
authors.forename: Laurids Siig
authors.initials: LS
authors.lastname: Christensen
authors.forename: Birgit
authors.initials: B
authors.lastname: Nørrung
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Hoorfar
authors.forename: Jan
authors.initials: J
authors.lastname: Vinjé
mesh_terms: D017250:Caliciviridae Infections / Q000175:diagnosis* / Q000821:virology*; D019411:Clinical Laboratory Techniques / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D006801:Humans; D029322:Norovirus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D016366:Open Reading Frames; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014773:Virology / Q000379:methods*
chemical_list: D017931:DNA Primers