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

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 realtime 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 ≤1.5 TCID 50 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 TCID 50 (p < 0.001, R 2 = 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.

Sub-clinical infection of farmed Atlantic salmon Salmo salar with salmonid alphavirus— a prospective longitudinal study

“…In a previous study (Graham et al 2006), RRT-PCR was found to be more sensitive than screening for viraemia, and the same observation was made here (Fig. 1).…”

“…A sample size of 20 gives 95% confidence of detecting 15% prevalence. The failure to detect infection before this (by viraemia, RRT-PCR or serology) suggests that it was present at very low levels and negative results can be attributed to sampling error.In a previous study (Graham et al 2006), RRT-PCR was found to be more sensitive than screening for viraemia, and the same observation was made here (Fig. 1).…”

“…RNA extraction from sera and heart, and subsequent testing by RRT-PCR was conducted as previously described (Graham et al 2006). Briefly, RNA was extracted from 200 µl of sera using the High Pure Viral RNA kit (Roche) and RNA extraction from heart tissue was performed using the QIAGEN RNeasy Mini Kit (Qiagen).…”

Transcriptomic analysis of the host response to early stage salmonid alphavirus (SAV-1) infection in Atlantic salmon Salmo salar L.

“…Quantification of viral gene expression and validation of differential expression of host genes of the kidney tissue by qRT-PCR Quantitative real-time PCR (qRT-PCR) targeted the viral transmembrane glycoprotein [19] and a subset of key host genes (n ¼ 8) that were found to be significantly differentially expressed by microarray analysis, along with three reference 'housekeeping' genes, translation elongation factor 1 a (ELF 1a), b-actin, and cofilin-2 were also subject to qRT-PCR. PCR primer sets are detailed in Table 1 and were designed from EST sequences corresponding to the relevant TRAITS/SGP microarray features save for the virus primers.…”

Lack of evidence for vertical transmission of SAV 3 using gametes of Atlantic salmon, Salmo salar L., exposed by natural and experimental routes

“…SAV 1 was detected by PCR in the hearts of experimentally infected fish from 7 days post‐i.p. injection with cell‐cultured virus (Graham, Taylor, Rodgers, Weston, Khalili, Ball, Christie & Todd 2006; Christie, Graham, McLoughlin, Villoing, Todd & Knappskog 2007), and in serum from 3 days post‐challenge (p.c.) (McLoughlin, Graham, Norris, Matthews, Foyle, Rowley, Jewhurst, MacPhee & Todd 2006).…”