title: A newly developed BVDV-1 RT-qPCR Taqman assay based on Italian isolates: evaluation as a diagnostic tool.
abstract: A single-step TaqMan® RT-qPCR was developed for the detection of bovine viral diarrhea virus type 1 (BVDV-1), an important pathogen of cattle worldwide. The assay was based on conserved 5'UTR sequences of Italian BVDV-1 isolates. In order to establish a diagnostic protocol which simplifies sample collection and processing, the assay was tested on a variety of biological specimens collected from persistently infected calves. The samples analyzed included PBMCs, plasma, dry blood, ear notch and hair bulb. Time and costs required for the analysis of each type of specimen were compared. The RT-qPCR, whose lower limit of detection was 100 copies of viral RNA (1 TCID50), correctly identified all PI animals, irrespective of the type of specimen. The highest copy numbers were obtained from the RNAs extracted from PBMCs, ear notches and hair bulbs. Hair bulb-supernatants directly used as a template allowed identification of all PI animals. In conclusion, based on time and cost evaluation, the most effective and efficient protocol was the one based on the direct analysis of hair bulb-supernatants, avoiding the RNA extraction step.
authors.affiliation: Department of Veterinary Sciences, University of Pisa, 56124, Pisa, Italy.
authors.forename: Roberto
authors.initials: R
authors.lastname: Zoccola
authors.affiliation: Department of Veterinary Sciences, University of Pisa, 56124, Pisa, Italy.
authors.forename: Maurizio
authors.initials: M
authors.lastname: Mazzei
authors.affiliation: Scuola Normale Superiore, 56126, Pisa, Italy.
authors.forename: Maria Luisa
authors.initials: ML
authors.lastname: Carrozza
authors.affiliation: Department of Veterinary Sciences, University of Pisa, 56124, Pisa, Italy.|Veterinary Pathology, School of Veterinary Science, University of Liverpool, Neston, UK.
authors.forename: Emanuele
authors.initials: E
authors.lastname: Ricci
authors.affiliation: Department of Veterinary Sciences, University of Pisa, 56124, Pisa, Italy.
authors.forename: Mario
authors.initials: M
authors.lastname: Forzan
authors.affiliation: Department of Veterinary Sciences, University of Pisa, 56124, Pisa, Italy. federica.pizzurro@for.unipi.it.
authors.forename: Federica
authors.initials: F
authors.lastname: Pizzurro
authors.affiliation: Istituto Zooprofilattico Sperimentale dell'Umbria e delle Marche, via Salvemini 1, 06126, Perugia, Italy.
authors.forename: Monica
authors.initials: M
authors.lastname: Giammarioli
authors.affiliation: Department of Veterinary Sciences, University of Pisa, 56124, Pisa, Italy.
authors.forename: Patrizia
authors.initials: P
authors.lastname: Bandecchi
authors.affiliation: Department of Veterinary Sciences, University of Pisa, 56124, Pisa, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Tolari
mesh_terms: D000818:Animals; D001912:Bovine Virus Diarrhea-Mucosal Disease / Q000175:diagnosis* / Q000821:virology*; D002417:Cattle; D030241:Diarrhea Virus 1, Bovine Viral / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D007558:Italy; D012367:RNA, Viral / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D019914:Taq Polymerase / Q000235:genetics / Q000378:metabolism
chemical_list: D012367:RNA, Viral; D019914:Taq Polymerase
keywords: Bovine viral diarrhea; Ear notch; Hair bulb; Italian genotype; Persistently infected; Real time PCR