Development and evaluation of novel one-step TaqMan realtime RT-PCR assays for the detection and direct genotyping of genogroup I and II noroviruses

TY - JOUR

T1 - Development and evaluation of novel one-step TaqMan realtime RT-PCR assays for the detection and direct genotyping of genogroup I and II noroviruses

AU - Schultz, Anna Charlotte

AU - Varga, Everardo

AU - Dalsgaard, Anders

AU - Christensen, Laurids Siig

AU - Nørrung, Birgit

AU - Hoorfar, Jeffrey

AU - Vinjé, Jan

PY - 2011/3

Y1 - 2011/3

N2 - 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. Study design: 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.

AB - 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. Study design: 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.

KW - Former LIFE faculty

KW - Norovirus

KW - Detection

KW - Genotyping

KW - Realtime RT-PCR assay

U2 - 10.1016/j.jcv.2010.12.001

DO - 10.1016/j.jcv.2010.12.001

M3 - Journal article

C2 - 21195660

SN - 1386-6532

VL - 50

SP - 230

EP - 234

JO - Journal of Clinical Virology

JF - Journal of Clinical Virology

IS - 3

ER -