title: Use of reverse transcription-real time polymerase chain reaction (real time RT-PCR) assays with Universal Probe Library (UPL) probes for the detection and genotyping of infectious pancreatic necrosis virus strains isolated in Chile.
abstract: Reverse transcription-real time polymerase chain reaction (real time RT-PCR) assay with Universal Probe Library (UPL) probes has been developed for the detection and genotyping of Chilean infectious pancreatic necrosis virus (IPNV) isolates from infected cell culture. Partial nucleotide sequences (1175 bp) of the VP2 coding region from a selection of 7 Chilean IPNV isolates showed that they clustered into two main groups strongly correlated with Genogroups 1 and 5 proposed by Blake et al. (2001), corresponding to types West Buxton (WB) and Spajarup (Sp), respectively. Based on the VP2 gene sequences of those 7 Chilean isolates and different reference IPNV strains, 2 sets of candidate primer/UPL probes (# 8 and # 117) were designed and evaluated with a total of 32 field isolates isolated from Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss) and Pacific salmon (Oncorhynchus kisutch) farms from 2006 to 2010 in Chile. The UPL probes clearly differentiated the same two major Genogroups that those recognized by sequencing analysis. Among the Chilean isolates examined, 18 yielded amplification with UPL probe # 8, and 14 with probe # 117, respectively corresponding to types Sp and WB, as demonstrated by typing by sequencing. Based on the findings reported below, it has been demonstrated that the combined real time RT-PCR protocol with UPLs approach was efficient in discriminating distinct Genogroups of IPNV cultured in fish cell lines and, therefore, recommended its use for detection and typing of IPN viruses. The study also confirmed the existence of two IPNV type strains in Chilean salmonid aquaculture.
authors.affiliation: Laboratorio de Patología de Organismos Acuáticos y Biotecnología Acuícola, Facultad de Ciencias Biológicas, Universidad Andrés Bello, Viña del Mar, Chile.
authors.forename: Felipe
authors.initials: F
authors.lastname: Calleja
authors.forename: Marcos G
authors.initials: MG
authors.lastname: Godoy
authors.forename: Juan G
authors.initials: JG
authors.lastname: Cárcamo
authors.forename: Isabel
authors.initials: I
authors.lastname: Bandín
authors.forename: Alejandro J
authors.initials: AJ
authors.lastname: Yáñez
authors.forename: Carlos P
authors.initials: CP
authors.lastname: Dopazo
authors.forename: Fred S
authors.initials: FS
authors.lastname: Kibenge
authors.forename: Ruben
authors.initials: R
authors.lastname: Avendaño-Herrera
mesh_terms: D000818:Animals; D002677:Chile; D005393:Fish Diseases / Q000821:virology; D005838:Genotype; D018061:Infectious pancreatic necrosis virus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000235:genetics*; D017684:Oncorhynchus kisutch / Q000821:virology; D017686:Oncorhynchus mykiss / Q000821:virology; D012367:RNA, Viral / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D020317:Salmo salar / Q000821:virology; D017422:Sequence Analysis, DNA; D014730:Veterinary Medicine / Q000379:methods; D014773:Virology / Q000379:methods*
chemical_list: D015345:Oligonucleotide Probes; D012367:RNA, Viral