Comparative evaluation of five serological methods and RT-PCR assay for the detection of IPNV in fish

In the present study, six diagnostic methods for the detection of infectious pancreatic necrosis virus (IPNV) (indirect immunofluorescence, flow cytometry, immunoperoxidase, immunodot blot, immunostaphylococcus-protein A, and RT-PCR) have been comparatively evaluated using the seroneutralization as the reference assay, and 83 Spanish isolates and 3 reference strains. The most reliable methods were flow cytometry and RT-PCR which could detect virus at titers of 1 × 102 and 1 × 103 TCID50/ml, respectively. At a multiplicity of infection of 50, both assays allowed the earliest detection of IPNV at 4 h post-inoculation. Indirect immunofluorescence and immunoperoxidase assays required at least 6 h post-inoculation to detect viral antigens. The immunodot blot assay possesses low sensitivity and the immunostaphylococcus-protein A test cannot be applied for routine examination of IPNV. Positive reactions were obtained in 100% of the samples tested by seroneutralization and RT-PCR, 90.4% by the flow cytometry, 80.7% by the indirect immunofluorescence assay, 67.5% by the immunoperoxidase, 62.6% by the immunodot blot, and only 27.7% by immunostaphylococcus-protein A test. Therefore, RT-PCR and flow cytometry were the most appropriate and sensitive methods for the routine detection of IPNV from affected fish.This study was supported by grants No. AGF98-0837 and MAR95-1949-CO2 from the C.I.C.y T. (Ministerio de Educación y Ciencia, Spanish Government), and 98-07B/0023 from the Comunidad de Madrid. We would also like to thank Laboratorio de Quı́mica de Proteı́nas del Centro de Investigaciones Biológicas (C.I.B.), C.S.I.C, Madrid for the synthesis of the oligonucleotide primers, and M.J. Navarrete for her help in the English review