title: Development and validation of a reverse transcription quantitative PCR for universal detection of viral hemorrhagic septicemia virus.
abstract: Viral hemorrhagic septicemia virus (VHSV) infects over 70 fish species inhabiting marine, brackish or freshwater environments throughout the Northern Hemisphere. Over its geographic range, 4 VHSV genotypes and multiple subtypes exist. Here, we describe the development and validation of a rapid, sensitive and specific real-time reverse transcription quantitative PCR assay (RT-qPCR) that amplifies sequence from representative isolates of all VHSV genotypes (I, II, III and IV). The pan-specific VHSV RT-qPCR assay reliably detects 100 copies of VHSV nucleoprotein RNA without cross-reacting with infectious hematopoietic necrosis virus, spring viremia of carp virus or aquatic birnavirus. Test performance characteristics evaluated on experimentally infected Atlantic salmon Salmo salar L. revealed a diagnostic sensitivity (DSe) > or = 93% and specificity (DSp) = 100%. The repeatability and reproducibility of the procedure was exceptionally high, with 93% agreement among test results within and between 2 laboratories. Furthermore, proficiency testing demonstrated the VHSV RT-qPCR assay to be easily transferred to and performed by a total of 9 technicians representing 4 laboratories in 2 countries. The assay performed equivalent to the traditional detection method of virus isolation via cell culture with the advantage of faster turnaround times and high throughput capacity, further suggesting the suitability of the use of this VHSV RT-qPCR in a diagnostic setting.
authors.affiliation: Fisheries & Oceans Canada, Pacific Biological Station, Nanaimo, British Columbia, V9T 6N7, Canada. kyle.garver@dfo-mpo.gc.ca
authors.forename: Kyle A
authors.initials: KA
authors.lastname: Garver
authors.forename: Laura M
authors.initials: LM
authors.lastname: Hawley
authors.forename: Carol A
authors.initials: CA
authors.lastname: McClure
authors.forename: Tamara
authors.initials: T
authors.lastname: Schroeder
authors.forename: Sandra
authors.initials: S
authors.lastname: Aldous
authors.forename: Fiona
authors.initials: F
authors.lastname: Doig
authors.forename: Michael
authors.initials: M
authors.lastname: Snow
authors.forename: Sandra
authors.initials: S
authors.lastname: Edes
authors.forename: Catherine
authors.initials: C
authors.lastname: Baynes
authors.forename: Jon
authors.initials: J
authors.lastname: Richard
mesh_terms: D000818:Animals; D001483:Base Sequence; D005838:Genotype; D031941:Hemorrhagic Septicemia, Viral / Q000175:diagnosis* / Q000821:virology; D029141:Novirhabdovirus / Q000235:genetics / Q000302:isolation & purification*; D012367:RNA, Viral / Q000235:genetics; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012474:Salmon; D012680:Sensitivity and Specificity
chemical_list: D012367:RNA, Viral