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

Diseases of Aquatic Organisms

C. Taylor D. Rodgers J. Weston M. Khalili N. Ball K. E. Christie D. Todd¹  D. A. Graham¹ 

关键词: Real-time RT-PCR;    SYBR green;    Salmonid alphavirus;    Serum;    Heart;    Diagnosis;   

DOI  :  10.3354/dao070047

学科分类：生物科学（综合）

来源: Inter-Research

【 摘 要 】

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.

【 授权许可】

Unknown