title: 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 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.
authors.affiliation: Department of Agriculture and Rural Development, Veterinary Sciences Division, Stoney Road, Stormont, Belfast BT4 3SD, UK. david.graham@dardni.gov.uk
authors.forename: D A
authors.initials: DA
authors.lastname: Graham
authors.forename: C
authors.initials: C
authors.lastname: Taylor
authors.forename: D
authors.initials: D
authors.lastname: Rodgers
authors.forename: J
authors.initials: J
authors.lastname: Weston
authors.forename: M
authors.initials: M
authors.lastname: Khalili
authors.forename: N
authors.initials: N
authors.lastname: Ball
authors.forename: K E
authors.initials: KE
authors.lastname: Christie
authors.forename: D
authors.initials: D
authors.lastname: Todd
mesh_terms: D000524:Alphavirus / Q000235:genetics / Q000302:isolation & purification*; D018354:Alphavirus Infections / Q000097:blood / Q000662:veterinary*; D000818:Animals; D017931:DNA Primers / Q000737:chemistry; D005393:Fish Diseases / Q000097:blood / Q000821:virology*; D006321:Heart / Q000821:virology; D012367:RNA, Viral / Q000032:analysis; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012487:Salmonidae / Q000821:virology*; D013997:Time Factors; D019562:Viral Load / Q000662:veterinary; D014764:Viral Proteins / Q000235:genetics
chemical_list: D017931:DNA Primers; D012367:RNA, Viral; D014764:Viral Proteins