Accurate strand-specific quantification of viral RNA
Autor(es): Plaskon Nicole E, Adelman Zach N, Myles Kevin M
Resumo: 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 - measuring specific str-s of viral RNA in infected cells - tissues are important in the study of RNA viruses. Str--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 str- present in the RT reaction. To specifically test this assumption, we developed multiple ssqPCR assays for the positive-str- 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 st-ard unmodified virus-specific primers failed to discern the difference between cDNAs generated from virus specific primers - those generated through false priming. Further, we were unable to accurately measure levels of ONNV (-) str- RNA with this assay when higher levels of cDNA generated from the (+) str- were present. Taken together, these results suggest that assays of this type do not accurately quantify levels of the anti-genomic str- 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 (-) str- RNA from other cDNAs present, thus allowing accurate quantification of the anti-genomic str-. We also report the sensitivities of two different detection strategies - chemistries, SYBR(R) Green - DNA hydrolysis probes, used with our tagged ONNV-specific ssqPCR assays. Finally, we describe development, design - validation of ssqPCR assays for chikungunya virus (CHIKV), the recent cause of large outbreaks of disease in the Indian Ocean region.
Imprenta: PloS One, v. 4, n. 10, p. e7468, 2009
Identificador do objeto digital: 10.1371/journal.pone.0007468
Descritores: Chikungunya virus - Biosynthesis ; Chikungunya virus - Cell ; Chikungunya virus - DNA ; Chikungunya virus - Pathogenesis ; Chikungunya virus - RNA ; Chikungunya virus - PCR detection ; Chikungunya virus - Real Time PCR ; Chikungunya virus - RT-PCR ; Chikungunya Virus - Virus ; Chikungunya virus - Public health
Data de publicação: 2009
