A highly sensitive strand-specific multiplex RT-qPCR assay for quantitation of Zika virus replication

Abstract

Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) is widely used to quantify viral RNA genomes for diagnostics and research, yet conventional RT-qPCR protocols are unable to accurately distinguish between the different viral RNA species that exist during infection. Here we show that false-priming and self-priming occur during reverse transcription with several published Zika virus (ZIKV) primer sets. We developed a RT-qPCR assay using tagged primers and thermostable reverse transcriptase, which greatly reduced the occurrence of nonspecific cDNA products. Furthermore, we optimized the assay for use in multiplex qPCR which allows for simultaneous quantitative detection of positive-strand and negative-strand ZIKV RNA along with an internal control from both human and mosquito cells. Importantly, this assay is sensitive enough to study early stages of virus infection in vitro. Strikingly, using this assay, we detected ZIKV negative-strand RNA as early as 3 h post-infection in mammalian cell culture, at a time point prior to the onset of positive-strand RNA synthesis. Overall, the strand-specific RT-qPCR assay developed herein is a valuable tool to quantify ZIKV RNA and to study viral replication dynamics during infection. The application of these findings has the potential to increase accuracy of RNA detection methods for a variety of viral pathogens.HighlightsSelf-primed cDNA is amplified by widely-used ZIKV qPCR primer setsUse of tagged primers and thermostable RT increases strand-specificity for RT-qPCRMultiplexed qPCR allows for simultaneous quantitation of (+) and (-) strand viral RNAs, and an internal controlStrand-specific RT-qPCR can detect fewer than one copy of viral RNA per cell in human and mosquito cells