Design, development, and validation of a strand-specific RT-qPCR assay for GI and GII human Noroviruses.

cam.issuedOnline2021-09-23
dc.contributor.authorKönig, Katja Marie Kjara
dc.contributor.authorJahun, Aminu S
dc.contributor.authorNayak, Komal
dc.contributor.authorDrumright, Lydia N
dc.contributor.authorZibauer, Matthias
dc.contributor.authorGoodfellow, Ian
dc.contributor.authorHosmillo, Myra
dc.contributor.orcidJahun, Aminu S [0000-0002-4585-1701]
dc.contributor.orcidGoodfellow, Ian [0000-0002-9483-510X]
dc.contributor.orcidHosmillo, Myra [0000-0002-3514-7681]
dc.date.accessioned2022-01-06T12:56:22Z
dc.date.available2022-01-06T12:56:22Z
dc.date.issued2021
dc.date.updated2022-01-06T12:56:20Z
dc.description.abstractHuman noroviruses (HuNoV) are the major cause of viral gastroenteritis worldwide. Similar to other positive-sense single-stranded RNA viruses, norovirus RNA replication requires the formation of a negative strand RNA intermediate. Methods for detecting and quantifying the viral positive or negative sense RNA in infected cells and tissues can be used as important tools in dissecting virus replication. In this study, we have established a sensitive and strand-specific Taqman-based quantitative polymerase chain reaction (qPCR) assay for both genogroups GI and GII HuNoV. This assay shows good reproducibility, has a broad dynamic range and is able to detect a diverse range of isolates. We used tagged primers containing a non-viral sequence for the reverse transcription (RT) reaction and targeted this tag in the succeeding qPCR reaction to achieve strand specificity. The specificity of the assay was confirmed by the detection of specific viral RNA strands in the presence of high levels of the opposing strands, in both RT and qPCR reactions. Finally, we further validated the assay in norovirus replicon-bearing cell lines and norovirus-infected human small intestinal organoids, in the presence or absence of small-molecule inhibitors. Overall, we have established a strand-specific qPCR assay that can be used as a reliable method to understand the molecular details of the human norovirus life cycle.
dc.identifier.doi10.17863/CAM.79667
dc.identifier.eissn2398-502X
dc.identifier.issn2398-502X
dc.identifier.otherPMC8506223
dc.identifier.other34708158
dc.identifier.urihttps://www.repository.cam.ac.uk/handle/1810/332221
dc.languageeng
dc.publisherF1000 Research Ltd
dc.publisher.urlhttp://dx.doi.org/10.12688/wellcomeopenres.17078.1
dc.rightsAttribution 4.0 International
dc.rights.urihttps://creativecommons.org/licenses/by/4.0/
dc.sourceessn: 2398-502X
dc.sourcenlmid: 101696457
dc.subjectHuNoV replicon
dc.subjectRT-qPCR
dc.subjectcalicivirus
dc.subjecthuman intestinal organoids
dc.subjecthuman norovirus
dc.subjectintestinal epithelial cells
dc.subjectstrand-specific RT-qPCR
dc.subjectviral gastroenteritis
dc.titleDesign, development, and validation of a strand-specific RT-qPCR assay for GI and GII human Noroviruses.
dc.typeArticle
dcterms.dateAccepted2021-09-15
prism.publicationNameWellcome Open Res
prism.volume6
pubs.funder-project-idWellcome Trust (207498/Z/17/Z)
pubs.funder-project-idMRC (MR/T001917/1)
rioxxterms.licenseref.urihttps://creativecommons.org/licenses/by/4.0/
rioxxterms.versionVoR
rioxxterms.versionofrecord10.12688/wellcomeopenres.17078.1
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