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Development and validation of a real-time TaqMan PCR assay for the detection of betanodavirus in clinical specimens

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Abstract

Betanodaviruses are the causal agents of viral encephalo-retinopathy, an infectious disease affecting more than 40 marine fish species, characterized by high morbidity and mortality. Because of its severe impact, robust diagnostic tools are required. The aim of this work was to develop and validate a real-time TaqMan PCR assay to detect betanodaviruses in clinical specimens by amplifying a conserved region of the RNA2 strand. The method proved to be specific and sensitive, being capable of detecting as low as 10 TCID50/ml. For clinical validation, samples from 100 marine fish were collected during a natural outbreak of disease and tested by three distinct laboratory methods, namely real-time TaqMan PCR, RT-seminested PCR and virus isolation. The results indicated optimal agreement between tests. The assay that was developed is capable of detecting members of all of the betanodavirus genetic groups currently described and can be considered a valid alternative to the time-consuming and contamination-prone nested PCR.

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Acknowledgments

This study was supported by the Italian Ministry of Health (RC IZSVe 02/06). The authors thank Dr. Cristian De Battisti for the technical advice (IZSVe), Dr. Marzia Mancin for the statistical analysis (IZSVe), Dr. William Dundon for critical editing of the manuscript (IZSVe) and Dr. Hilde Sindre (National Veterinary Institute, Norway) for providing betanodavirus isolates of Atlantic origin.

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Authors and Affiliations

  1. Fish and Shellfish Pathology Department, OIE Reference Laboratory for Viral Encephalopathy and Retinopathy, Istituto Zooprofilattico Sperimentale delle Venezie, Viale dell’Università 10, 35020, Legnaro, Padova, Italy

    V. Panzarin, P. Patarnello, A. Mori, E. Rampazzo, E. Cappellozza, G. Bovo & G. Cattoli

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  1. V. Panzarin
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  2. P. Patarnello
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  3. A. Mori
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  4. E. Rampazzo
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  5. E. Cappellozza
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  6. G. Bovo
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  7. G. Cattoli
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Correspondence to V. Panzarin.

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Panzarin, V., Patarnello, P., Mori, A. et al. Development and validation of a real-time TaqMan PCR assay for the detection of betanodavirus in clinical specimens. Arch Virol 155, 1193–1203 (2010). https://doi.org/10.1007/s00705-010-0701-5

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  • DOI: https://doi.org/10.1007/s00705-010-0701-5

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