Development and validation of a real-time TaqMan PCR assay for the detection of betanodavirus in clinical specimens.
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 TCID(50)/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.
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. vpanzarin@izsvenezie.it
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Panzarin
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Patarnello
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Mori
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Rampazzo
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Cappellozza
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Bovo
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Cattoli
D000818:Animals; D005393:Fish Diseases / Q000175:diagnosis* / Q000821:virology; D030381:Nodaviridae / Q000235:genetics / Q000302:isolation & purification*; D010473:Perciformes / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods; D012327:RNA Virus Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D012367:RNA, Viral / Q000032:analysis; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D019914:Taq Polymerase
D012367:RNA, Viral; D019914:Taq Polymerase