Development of a strand-specific real-time qRT-PCR for the accurate detection and quantitation of West Nile virus RNA
作者： Stephanie M. Lim , Penelope Koraka , Albert D.M.E. Osterhaus , Byron E.E. Martina
DOI: 10.1016/J.JVIROMET.2013.07.050
关键词:
摘要: Studying the tropism and replication kinetics of West Nile virus (WNV) in different cell types vitro tissues animal models is important for understanding its pathogenesis. As detection negative strand viral RNA a more reliable indicator active single-stranded positive-sense viruses, specificity qRT-PCR assays currently used WNV positive was reassessed. It shown that self- falsely-primed cDNA generated during reverse transcription step an assay employing unmodified primers several transcriptases. result, using thermostable rTth combination with tagged developed, which greatly improved by circumventing events false-priming. The reliability then addressed BV-2 microglia cells as well C57/BL6 mice. possible to follow negative-strand synthesis both vivo; however, sensitivity will need be optimized order detect quantify very early stages infection. Overall, strand-specific developed this study effective tool RNA, reassess replication, context
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