title: Design and validation of a RT-qPCR procedure for diagnosis and quantification of most types of infectious pancreatic necrosis virus using a single pair of degenerated primers.
abstract: Infectious pancreatic necrosis virus (IPNV) is an important virus which affects the salmonid aquaculture industry worldwide; therefore, it is important to develop rapid and reliable methods of diagnosis to detect the disease at early stages. Nowadays, RT-qPCR is replacing other methods because it provides additional information on the viral load, which is important to have a better understanding of the virus replication level and of the stage of the infection and its risk level. The main problem stems from the high diversity of this virus, which can compromise the reliability of the diagnosis. In this study, we have designed an RT-qPCR procedure for diagnosis and quantification of IPNV based on a single pair of primers targeted to segment B. The procedure has been validated, in vitro and in vivo, testing two different types of standards against seven reference strains and 23 field isolates from different types. The procedure is reliable for the detection of any type, with a detection limit of 31 TCID50mL-1, 50 pfu mL-1or 66 RNA copies mL-1, depending on the standard. All the standard curves showed high reliability (R2> 0.95). The results support the high reliability of this new procedure for the diagnosis and quantification of IPNV.
authors.affiliation: Departamento de Microbiología y Parasitología, Instituto de Acuicultura, Universidade de Santiago de Compostela, Santiago de Compostela, Spain.
authors.forename: D
authors.initials: D
authors.lastname: Vázquez
authors.affiliation: Departamento de Microbiología y Parasitología, Instituto de Acuicultura, Universidade de Santiago de Compostela, Santiago de Compostela, Spain.
authors.forename: J M
authors.initials: JM
authors.lastname: Cutrín
authors.affiliation: Departamento de Microbiología y Parasitología, Instituto de Acuicultura, Universidade de Santiago de Compostela, Santiago de Compostela, Spain.
authors.forename: J G
authors.initials: JG
authors.lastname: Olveira
authors.affiliation: Departamento de Microbiología y Parasitología, Instituto de Acuicultura, Universidade de Santiago de Compostela, Santiago de Compostela, Spain.
authors.forename: C P
authors.initials: CP
authors.lastname: Dopazo
mesh_terms: D000818:Animals; D018175:Birnaviridae Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D005393:Fish Diseases / Q000175:diagnosis* / Q000821:virology; D055772:Green Chemistry Technology; D018061:Infectious pancreatic necrosis virus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D010473:Perciformes; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012474:Salmon
chemical_list: D017931:DNA Primers
keywords: 
  IPNV
  ; RT-qPCR; diagnosis; quantification