一种针对传染性鲑鱼贫血病毒（ISAV）复制 RNA 的三种物种（vRNA、cRNA 和 mRNA）的靶向检测的单链特异性实时 RT-PCR 方法。

A strand specific real-time RT-PCR method for the targeted detection of the three species (vRNA, cRNA and mRNA) of infectious salmon anaemia virus (ISAV) replicative RNA.

机构信息

Marine Scotland Science, Marine Laboratory, 375 Victoria Road, Aberdeen, Scotland, UK.

出版信息

J Virol Methods. 2013 Jan;187(1):65-71. doi: 10.1016/j.jviromet.2012.09.007. Epub 2012 Sep 13.

DOI:10.1016/j.jviromet.2012.09.007

PMID:22982076

Abstract

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.

摘要

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。