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Diseases of Aquatic Organisms

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DAO 72:107-113 (2006)  -  doi:10.3354/dao072107

Detection of infectious salmon anaemia virus by real-time nucleic acid sequence based amplification

William G. Starkey1,*, David A. Smail2, Hogne Bleie3, K. Fiona Muir1, Jacqueline H. Ireland1, Randolph H. Richards1

1Department of Virology, Institute of Aquaculture, University of Stirling, Stirling FK9 4LA, UK
2F.R.S. Marine Laboratory, PO Box 101, Aberdeen AB11 9DB, UK
3National Veterinary Institute, PO Box 1263, Sentrum, 5811 Bergen, Norway

ABSTRACT: We have developed a real-time nucleic acid sequence based amplification (NASBA) procedure for detection of infectious salmon anaemia virus (ISAV). Primers were designed to target a 124 nucleotide region of ISAV genome segment 8. Amplification products were detected in real-time with a molecular beacon (carboxyfluorescin [FAM]-labelled and methyl-red quenched) that recognised an internal region of the target amplicon. Amplification and detection were performed at 41°C for 90 min in a Corbett Research Rotorgene. The real-time NASBA assay was compared to a conventional RT-PCR for ISAV detection. From a panel of 45 clinical samples, both assays detected ISAV in the same 19 samples. Based on the detection of a synthetic RNA target, the real-time NASBA procedure was approximately 100× more sensitive than conventional RT-PCR. These results suggest that real-time NASBA may represent a useful diagnostic procedure for ISAV.

KEY WORDS: Infectious salmon anaemia virus · Orthomyxovirus · NASBA · Diagnostics · Fish · Nucleic acid amplification

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