Accurate strand-specific quantification of viral RNA

Author(s): Plaskon NE, Adelman ZN, Myles KM

Abstract

The 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(R) 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.

Similar Articles

Potato spindle tuber "virus"

Author(s): Diener TO

Inhibition of potato spindle tuber viroid (PSTVd) infection with DNA oligonucleotides

Author(s): Matousek J, Trnena L, Arnold L, Dedic P, Rakousky S, et al.

Behavior of a hammerhead ribozyme in aqueous solution at medium to high temperatures

Author(s): El-Murr N, Maurel MC, Rihova M, Vergne J, Hervé G, et al.

The revised classification of eukaryotes

Author(s): Adl SM, Simpson AG, Lane CE, Lukes J, Bass D, et al.

Improving the coverage of the cyanobacterial phylum using diversity-driven genome sequencing

Author(s): Shih PM, Wu D, Latifi A, Axen SD, Fewer DP, et al.

Self-cleavage of plus and minus RNA transcripts of avocado sunblotch viroid

Author(s): Hutchins CJ, Rathjen PD, Forster AC, Symons RH