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

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. Th...

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Bibliographic Details
Published in:Diseases of aquatic organisms 2006-06, Vol.70 (1-2), p.47-54
Main Authors: GRAHAM, D. A, TAYLOR, C, RODGERS, D, WESTON, J, KHALILI, M, BALL, N, CHRISTIE, K. E, TODD, D
Format: Article
Language:English
Subjects:
Alphavirus - genetics
Alphavirus - isolation & purification
Alphavirus Infections - blood
Alphavirus Infections - veterinary
Animal aquaculture
Animal productions
Animals
Biological and medical sciences
DNA Primers - chemistry
Fish Diseases - blood
Fish Diseases - virology
Fundamental and applied biological sciences. Psychology
Heart - virology
Marine
Pisciculture
Reproducibility of Results
Reverse Transcriptase Polymerase Chain Reaction - methods
Reverse Transcriptase Polymerase Chain Reaction - veterinary
RNA, Viral - analysis
Salmonid alphavirus
Salmonidae
Salmonidae - virology
Time Factors
Vertebrate aquaculture
Viral Load - veterinary
Viral Proteins - genetics
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Summary: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 10(7) dilution range. The limit of detection was calculated to be < or = 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.
ISSN:0177-5103
1616-1580
DOI:10.3354/dao070047