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

作者：McBeath Alastair^*; Bain Nicola; Fourrier Mickael; Collet Bertrand; Snow Michael

来源：Journal of Virological Methods, 2013, 187(1): 65-71.

DOI：10.1016/j.jviromet.2012.09.007

• ISAV
• RNA strand specific
• Real-time RT-PCR
• Replication

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 RI 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 degrees C), sub-optimal (20 degrees C) and inadequate (25 degrees 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 degrees C indicating the potential usefulness of the assay as a tool in the understanding of ISAV replication and transcription dynamics.

• 出版日期2013-1