Department of Microbiology & Immunology, McGill University, Montreal, QC, Canada.
Department of Microbiology & Immunology, McGill University, Montreal, QC, Canada; Department of Biochemistry, McGill University, Montreal, QC, Canada.
J Virol Methods. 2022 Sep;307:114556. doi: 10.1016/j.jviromet.2022.114556. Epub 2022 May 30.
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.
逆转录定量聚合酶链反应（RT-qPCR）广泛用于诊断和研究中定量检测病毒 RNA 基因组，但传统的 RT-qPCR 方案无法准确区分感染过程中存在的不同病毒 RNA 物种。在这里，我们展示了几种已发表的寨卡病毒（ZIKV）引物在逆转录过程中会发生错误引发和自我引发。我们使用标记引物和热稳定逆转录酶开发了一种 RT-qPCR 检测方法，极大地减少了非特异性 cDNA 产物的发生。此外，我们优化了该检测方法，使其可用于多重 qPCR，可同时定量检测正链和负链 ZIKV RNA 以及来自人和蚊子细胞的内部对照。重要的是，该检测方法足够灵敏，可用于研究体外病毒感染的早期阶段。值得注意的是，使用该检测方法，我们在哺乳动物细胞培养物中感染后 3 小时就检测到了 ZIKV 负链 RNA，此时正链 RNA 合成尚未开始。总的来说，本文开发的这种链特异性 RT-qPCR 检测方法是定量检测 ZIKV RNA 和研究感染过程中病毒复制动态的有力工具。这些发现的应用有可能提高各种病毒病原体 RNA 检测方法的准确性。
