Marine Scotland Science, Marine Laboratory, 375 Victoria Road, Aberdeen, Scotland, UK.
J Virol Methods. 2013 Jan;187(1):65-71. doi: 10.1016/j.jviromet.2012.09.007. Epub 2012 Sep 13.
Three species of viral-derived RNA (vRNA, cRNA and mRNA) are produced during an infectious salmon anaemia virus (ISAV) infection. Conventional real-time RT-PCR (RT-qPCR) targeting ISAV segment 8 provides a very sensitive method for the detection of ISAV RNA, however it does not differentiate between these three individual RNA species. In this study, strand-specific tagged primers have been utilised in the RT reaction to specifically produce cDNA corresponding to each of the 3 viral RNA types produced from ISAV segment 8 for the subsequent detection by real-time PCR. The RNA species-specific assay was successfully used to specifically distinguish synthetic T7-produced RNA transcripts representing the 3 species of ISAV RNA at levels up to approximately 10(5)-fold higher than the other types. In addition, the method was applied to investigate the production of segment 8 RNA in time-course tissue culture experiments performed at optimal (15°C), sub-optimal (20°C) and inadequate (25°C) temperatures for replication or in the presence of a chemical inhibitor to vary the RNA populations and investigate its effectiveness. Variation in RNA production was observed between the optimal and sub-optimal temperatures and in the presence of the chemical inhibitor. Production of all RNA species was completely inhibited at 25°C indicating the potential usefulness of the assay as a tool in the understanding of ISAV replication and transcription dynamics.
在传染性鲑鱼贫血病毒（ISAV）感染过程中会产生三种病毒衍生的 RNA（vRNA、cRNA 和 mRNA）。针对 ISAV 片段 8 的常规实时 RT-PCR（RT-qPCR）提供了一种非常敏感的方法来检测 ISAV RNA，但它不能区分这三种单独的 RNA 种类。在本研究中，在 RT 反应中使用了链特异性标记引物，以特异性地产生来自 ISAV 片段 8 的 3 种病毒 RNA 各自的 cDNA，随后通过实时 PCR 进行检测。该 RNA 种类特异性检测方法成功地用于特异性区分 T7 产生的 RNA 转录本，这些转录本代表 ISAV RNA 的 3 种 RNA 种类，其水平比其他类型高约 10（5）倍。此外，该方法还应用于研究在最佳（15°C）、次佳（20°C）和不适宜（25°C）温度下进行的组织培养实验中，以及在存在化学抑制剂以改变 RNA 群体的情况下，研究 ISAV 片段 8 RNA 的产生情况及其有效性。在最佳和次佳温度之间以及在化学抑制剂存在的情况下观察到 RNA 产生的变化。在 25°C 时，所有 RNA 种类的产生均被完全抑制，这表明该检测方法在理解 ISAV 复制和转录动力学方面具有潜在的用途。
