Absolute quantitation of infectious salmon anaemia virus using different real-time reverse transcription PCR chemistries

Routine laboratory diagnosis of infectious salmon anaemia virus (ISAV) infection is primarily by reverse transcription polymerase chain reaction (RT-PCR) because of the high sensitivity and rapid turnaround time of the test. This paper describes methods for highly reproducible absolute viral load measurements using external standard curves generated with either ISAV recombinant plasmid DNA (pDNA) standards or transcribed RNA standards prepared by in vitro transcription with T7 RNA polymerase, and using a two tube real-time or quantitative (q)RT-PCR with SYBR® Green I chemistry and a single tube qRT-PCR with TaqMan® probe chemistry. When applied to virus samples of known virus titer for the highly pathogenic ISAV strain NBISA01 and the low pathogenic ISAV strain RPC/NB-04-085-1, both methods showed a 100-fold lower detectable titer for RPC/NB-04-085-1 but with a higher number of viral RNA molecules compared to NBISA01. Overall, the SYBR® Green I method overestimated copy numbers in samples having equivalent Ct values with the TaqMan® probe method. Taken together, the findings suggest that the TaqMan® probe method with the in vitro transcribed RNA standard curve is the preferred method for reliable and rapid quantitation of ISAV in samples.