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Inter-Laboratory Ring Trial to Evaluate Reverse Transcription Polymerase Chain Reaction Methods Used for <i>Dolphin Morbillivirus</i>Detection in Italy

引用

Advances in Microbiology 2019年第5期9卷 423-437页

作者： Zoccola Roberto Pautasso Alessandra Grattarola Carla Monnier Milena Miceli Ilaria Ingravalle Francesco Crescio Maria Ines Giorda Federica Masoero Loretta zaccaria guendalina Purpari Giuseppa Cersini Antonella Viscardi Maurizio Serracca Laura Padalino Iolanda Puggioni Giantonella Canonico Cristina Toffan Anna Centelleghe Cinzia Di Francesco Cristina Esmeralda Mazzariol Sandro Di Guardo Giovanni Mignone Walter Casalone Cristina Goria Maria Istituto Zooprofilattico Sperimentale del Piemonte Liguria e Valle d’Aosta Torino Italy Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise Teramo Italy Istituto Zooprofilattico Sperimentale della Sicilia Palermo Italy Istituto Zooprofilattico Sperimentale del Lazio e della Toscana Roma Italy Istituto Zooprofilattico Sperimentale del Mezzogiorno Portici (NA) Italy Istituto Zooprofilattico Sperimentale del Piemonte Liguria e Valle d’Aosta La Spezia Italy Istituto Zooprofilattico Sperimentale della Puglia e Basilicata Foggia Italy Istituto Zooprofilattico Sperimentale della Sardegna Sassari Italy Istituto Zooprofilattico Sperimentale dell’ Umbria e delle Marche Ancona Italy Istituto Zooprofilattico Sperimentale delle Venezie Legnaro (PD) Italy University of Padova Department of Comparative Biomedicine and Food Science (BCA) Legnaro (PD) Italy Faculty of Veterinary Medicine University of Teramo Teramo Italy Istituto Zooprofilattico Sperimentale del Piemonte Liguria e Valle d’Aosta Imperia Liguria Italy

Dolphin Morbillivirus (DMV) is one of the most frequently detected pathogens in stranded cetacean specimens worldwide as well as in Italy. Due to the persistence of DMV in the Mediterranean Sea and to the lack of information about the efficiency of the available diagnostic techniques, the Italian National Reference Centre for diagnostic activities on dead stranded marine mammals (C.Re.Di.Ma) performed the first inter-laboratory ring trial with the aim to standardize a diagnostic biomolecular approach for DMV in Italy. Viral isolation is usually considered the “gold standard” for the definitive diagnosis of most pathogens, but it is not often feasible in DMV diagnosis, due to the poor preservation of virus-targeted tissues in stranded cetacean carcasses, as well as to the lack of appropriate sensitivity of cell lines towards DMV variability. Therefore direct viral detection on tissues by means of reverse transcription-PCR (RT-PCR) represents a valuable option for DMV infection’s diagnosis. For detecting DMV in cetacean die-offs occurred in the Mediterranean basin since 2013, C.Re.Di.Ma developed an RT-PCR based method targeting to a 287 bp fragment of DMV nucleoprotein (N) gene. With the purpose to evaluate its performances in terms of accuracy (Se = sensitivity and Sp = specificity) and precision (reproducibility), it was submitted to a ring trial. So, 12 Public Laboratories belonging to the Italian dead stranded marine mammals diagnostic network were asked to analyze a panel of 40 samples (positive and negative for DMV, using different dilutions of a viral suspension obtained from a cell culture supernatant of a DMV strain) with the aforementioned technique. Furthermore, we also aimed at comparing the accuracy of other 7 molecular methods routinely applied for DMV detection in Italy. For this purpose, the second panel of identical 40 DMV +ve and -ve samples was provided to Laboratories that routinely used DMV detection methods other than those developed by C.Re.Di.Ma, in order to be analyzed simultaneously with

关键词： Dolphin Morbillivirus RT-PCR Interlab Ring Test Method Comparison Stranded Cetaceans Mediterranean Sea

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