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

Anna Charlotte Schultz; Everardo Varga; Anders Dalsgaard; Laurids Siig Christensen; Birgit Nørrung; Jeffrey Hoorfar; Jan Vinjé

doi:10.1016/j.jcv.2010.12.001

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

Anna Charlotte Schultz, Everardo Varga, Anders Dalsgaard, Laurids Siig Christensen, Birgit Nørrung, Jeffrey Hoorfar, Jan Vinjé

16 Citations (Scopus)

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. 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.

Original language	English
Journal	Journal of Clinical Virology
Volume	50
Issue number	3
Pages (from-to)	230-234
Number of pages	5
ISSN	1386-6532
DOIs	https://doi.org/10.1016/j.jcv.2010.12.001
Publication status	Published - Mar 2011

Keywords

Former LIFE faculty

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10.1016/j.jcv.2010.12.001

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Development and evaluation of novel one-step TaqMan realtime RT-PCR assays for the detection and direct genotyping of genogroup I and II noroviruses. / Schultz, Anna Charlotte; Varga, Everardo; Dalsgaard, Anders et al.

In: Journal of Clinical Virology, Vol. 50, No. 3, 03.2011, p. 230-234.

Research output: Contribution to journal › Journal article › Research › peer-review

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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. 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.",

keywords = "Former LIFE faculty, Norovirus, Detection, Genotyping, Realtime RT-PCR assay",

author = "Schultz, {Anna Charlotte} and Everardo Varga and Anders Dalsgaard and Christensen, {Laurids Siig} and Birgit N{\o}rrung and Jeffrey Hoorfar and Jan Vinj{\'e}",

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doi = "10.1016/j.jcv.2010.12.001",

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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

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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

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