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

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• 作者：Alastair McBeath<sup> ; sup><sup>alastair.mcbeath@scotland.gsi.gov.uksup> ; Nicola Bain ; Mickael Fourrier ; Bertrand Collet ; Michael Snow<sup>1sup>
• 关键词：ISAV ; RNA strand specific ; Real-time RT-PCR ; Replication
• 刊名：Journal of Virological Methods
• 出版年：2013
• 出版时间：January, 2013
• 年：2013
• 卷：187
• 期：1
• 页码：65-71
• 全文大小：428 K

文摘

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<sup>5sup>-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 ¡ãC), sub-optimal (20 ¡ãC) and inadequate (25 ¡ãC) 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 ¡ãC indicating the potential usefulness of the assay as a tool in the understanding of ISAV replication and transcription dynamics.