title: Development and validation of a short-time cell culture and multiplex reverse transcriptase polymerase chain reaction assay for infectious pancreatic necrosis virus in Mexican farm-sampled rainbow trout.
abstract: The infectious pancreatic necrosis virus (IPNV) affects several species of freshwater and marine fish. In Mexico, IPNV has an important impact on farming of rainbow trout Oncorhynchus mykiss; however, IPNV distribution in Mexico is unclear. The diagnosis of IPNV is laborious; usually it is based on isolation tests in cell culture followed by immunological identification using techniques of serum neutralization, immunofluorescence, or enzyme-linked immunosorbent assay. It has recently been demonstrated that reverse transcriptase polymerase chain reaction (RT-PCR) is an adequate method for the detection of aquatic birnaviruses. However, its diagnostic use is still limited because very low titers of viable virus cannot be easily detected. In this study, a combination of short-time cell culture and multiplex RT-PCR was established for the diagnosis of IPNV in rainbow trout obtained from farms in the state of Mexico. Three primer sets were used in a single reaction in the multiplex RT-PCR to increase the probability of identifying all serotypes of IPNV serogroup A as well as to help prevent a false-negative result. This approach was able to identify samples with an IPNV concentration of just 0.01 tissue culture infective dose with 50% endpoint (TCID50)/mL, and it identified more infected fish than RT-PCR alone or first-passage cell culture alone. Moreover, this technique made the same identifications as second-passage cell culture but in approximately 30% of the time needed for second-passage cell culture. Consequently, the time and cost efficiency of IPNV diagnosis were greatly reduced.
authors.affiliation: Centro de Investigación y Estudios Avanzados en Salud Animal, Facultad de Medicina Veterinaria y Zootecnia, Universidad Autónoma del Estado de México, Carretera de Cuota Toluca-Atlacomulco, Kilómetro 15.5, Código Postal 50200, Toluca, Estado de México.
authors.forename: Magda
authors.initials: M
authors.lastname: Barrera-Mejía
authors.forename: J
authors.initials: J
authors.lastname: Simón-Martínez
authors.forename: Celene
authors.initials: C
authors.lastname: Salgado-Miranda
authors.forename: Fernando
authors.initials: F
authors.lastname: Vega
authors.forename: César
authors.initials: C
authors.lastname: Ortega
authors.forename: Andrés
authors.initials: A
authors.lastname: Aragón
mesh_terms: D000818:Animals; D018175:Birnaviridae Infections / Q000175:diagnosis / Q000453:epidemiology / Q000662:veterinary* / Q000821:virology; D018929:Cell Culture Techniques; D002460:Cell Line; D005393:Fish Diseases / Q000175:diagnosis / Q000453:epidemiology / Q000821:virology*; D018061:Infectious pancreatic necrosis virus; D008800:Mexico / Q000453:epidemiology; D017686:Oncorhynchus mykiss; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary*