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Diseases of Aquatic Organisms

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DAO 70:47-54 (2006)  -  doi:10.3354/dao070047

Development and evaluation of a one-step real-time reverse transcription polymerase chain reaction assay for the detection of salmonid alphaviruses in serum and tissues

D. A. Graham1,*, C. Taylor2, D. Rodgers1, J. Weston2, M. Khalili3, N. Ball1, K. E. Christie4, D. Todd1

1Department of Agriculture and Rural Development, Veterinary Sciences Division, Stoney Road, Stormont, Belfast BT4 3SD, UK
2Department of Veterinary Sciences, The Queen’s University of Belfast, Stoney Road, Stormont, Belfast BT4 3SD, UK
3Department of Pathobiology, Faculty of Veterinary Medicine, Shahid Bahonar University, 22 Bahman Boulevard, Kerman 76169-133, Iran
4Intervet Norbio, Thormohlensgt. 55, 5008 Bergen, Norway

ABSTRACT: We designed 4 primer pairs to amplify conserved regions of the E1 or nsP4 genes of salmonid alphavirus (SAV) and evaluated their performance in optimized 1-step SYBR green real-time RT-PCR (RRT-PCR) assays. A single primer pair, amplifying a 227 bp segment of E1 was then chosen for further study. This RRT-PCR was shown to be highly repeatable and reproducible over a wide range of RNA dilutions, with a linear relationship between cycle threshold (Ct) value and RNA concentration over a 107 dilution range. The limit of detection was calculated to be ≤1.5 TCID50 ml–1 . When applied to sera previously screened by virus isolation for SAV viraemia, the RRT-PCR correctly identified all 13 culture-positive samples, as well as finding an additional 28 sera positive. Relative semi-quantitation of sera showed a very highly significant relationship between copy number and TCID50 (p < 0.001, R2 = 0.9563). Following experimental infection of salmon, heart samples were consistently positive until 21 d post infection (dpi), with (weak) positive signals still detectable in 50% of fish 70 dpi.

KEY WORDS: Real-time RT-PCR · SYBR green · Salmonid alphavirus · Serum · Heart · Diagnosis

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