Accurate Strand-Specific Quantification of Viral RNA

Simple item page
dc.contributor.authorPlaskon, Nicole E.en
dc.contributor.authorAdelman, Zach N.en
dc.contributor.authorMyles, Kevin M.en
dc.contributor.departmentEntomologyen
dc.contributor.departmentFralin Life Sciences Instituteen
dc.date.accessioned2018-11-14T14:44:43Zen
dc.date.available2018-11-14T14:44:43Zen
dc.date.issued2009-10-22en
dc.description.abstractThe presence of full-length complements of viral genomic RNA is a hallmark of RNA virus replication within an infected cell. As such, methods for detecting and measuring specific strands of viral RNA in infected cells and tissues are important in the study of RNA viruses. Strand-specific quantitative real-time PCR (ssqPCR) assays are increasingly being used for this purpose, but the accuracy of these assays depends on the assumption that the amount of cDNA measured during the quantitative PCR (qPCR) step accurately reflects amounts of a specific viral RNA strand present in the RT reaction. To specifically test this assumption, we developed multiple ssqPCR assays for the positive-strand RNA virus o'nyong-nyong (ONNV) that were based upon the most prevalent ssqPCR assay design types in the literature. We then compared various parameters of the ONNV-specific assays. We found that an assay employing standard unmodified virus-specific primers failed to discern the difference between cDNAs generated from virus specific primers and those generated through false priming. Further, we were unable to accurately measure levels of ONNV (−) strand RNA with this assay when higher levels of cDNA generated from the (+) strand were present. Taken together, these results suggest that assays of this type do not accurately quantify levels of the anti-genomic strand present during RNA virus infectious cycles. However, an assay permitting the use of a tag-specific primer was able to distinguish cDNAs transcribed from ONNV (−) strand RNA from other cDNAs present, thus allowing accurate quantification of the anti-genomic strand. We also report the sensitivities of two different detection strategies and chemistries, SYBR® Green and DNA hydrolysis probes, used with our tagged ONNV-specific ssqPCR assays. Finally, we describe development, design and validation of ssqPCR assays for chikungunya virus (CHIKV), the recent cause of large outbreaks of disease in the Indian Ocean region.en
dc.description.versionPublished versionen
dc.format.mimetypeapplication/pdfen
dc.identifier.doihttps://doi.org/10.1371/journal.pone.0007468en
dc.identifier.eissn1932-6203en
dc.identifier.issue10en
dc.identifier.othere7468en
dc.identifier.pmid19847293en
dc.identifier.urihttp://hdl.handle.net/10919/85842en
dc.identifier.volume4en
dc.language.isoenen
dc.publisherPLOSen
dc.rightsCreative Commons Attribution 4.0 Internationalen
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/en
dc.titleAccurate Strand-Specific Quantification of Viral RNAen
dc.title.serialPLOS ONEen
dc.typeArticle - Refereeden
dc.type.dcmitypeTexten

Files

Original bundle
Now showing 1 - 1 of 1
Thumbnail Image
Name:
journal.pone.0007468.PDF
Size:
205.53 KB
Format:
Adobe Portable Document Format
Description:
Download

Collections

Scholarly Works, Fralin Life Sciences Institute