Sensitive and specific detection of Salmonid alphavirus using real-time PCR (TaqMan).
Pancreas disease is responsible for major economic losses in the European salmonid farming industry. It was previously believed that a single subtype (salmon pancreas disease virus) of the virus species Salmonid alphavirus (SAV) was responsible for all outbreaks of pancreas disease in the UK and Norway. However, the recent discovery that pancreas disease in Norway is caused by a new and distinct subtype of salmonid alphavirus, exclusively found in Norway, has advocated the need for better diagnostic tools. In the present paper, three real-time PCR assays for all known subtypes of salmonid alphavirus have been developed; the Q_nsP1 assay is a broad-spectrum one that detects RNA from all subtypes, the Q_SPDV assay specifically detects the salmon pancreas disease virus subtype, and the Q_NSAV assay only detects the new Norwegian salmonid alphavirus subtype. The results demonstrated the assays to be highly sensitive and specific, detecting <0.1TCID50 of virus stocks. Regression analysis and standard curves calculated from the Ct-values from 10-fold serial dilutions of virus stocks showed that the assays were highly reproducible over a wide range of RNA input. Thirty-nine field samples were tested in triplicate and compared with traditional RT-PCR. Overall, the real-time assays detected 35 positive compared to 29 positives in standard RT-PCR, and were thus shown to be more sensitive for detecting salmonid alphaviruses in field samples.