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

Wellcome Open Research

article

Katja Marie Kjara König¹  Aminu S. Jahun¹  Komal Nayak³  Lydia N. Drumright⁴  Matthias Zilbauer³  Ian Goodfellow¹  Myra Hosmillo¹ 

[1] Division of Virology, Department of Pathology, University of Cambridge, Addenbrooke's Hospital;Insitute of Chemistry and Metabolomics, Center for Structural and Cell Biology in Medicine ,(CSCM), University of Lübeck;Department of Paediatrics, University of Cambridge;Department of Medicine, University of Cambridge, Addenbrooke's Hospital;Department of Medicine, University of Washington

关键词: RT-qPCR;    strand-specific RT-qPCR;    human norovirus;    calicivirus;    human intestinal organoids;    intestinal epithelial cells;    HuNoV replicon;    viral gastroenteritis;   

DOI  :  10.12688/wellcomeopenres.17078.1

学科分类：内科医学

来源: Wellcome

【 摘 要 】

Human 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.

【 授权许可】

CC BY