title: Multiplex real-time RT-PCR assay for bovine viral diarrhea virus type 1, type 2 and HoBi-like pestivirus.
abstract: HoBi-like pestiviruses are emerging pestiviruses that infect cattle causing clinical forms overlapping to those induced by bovine viral diarrhea virus (BVDV) 1 and 2. As a consequence of their widespread distribution reported in recent years, molecular tools for rapid discrimination among pestiviruses infecting cattle are needed. The aim of the present study was to develop a multiplex real-time RT-PCR assay, based on the TaqMan technology, for the rapid and unambiguous characterisation of all bovine pestiviruses, including the emerging HoBi-like strains. The assay was found to be sensitive, specific and repeatable, ensuring detection of as few as 10(0)-10(1) viral RNA copies. No cross-reactions between different pestiviral species were observed even in samples artificially contaminated with more than one pestivirus. Analysis of field samples tested positive for BVDV-1, BVDV-2 or HoBi-like virus by a nested PCR protocol revealed that the developed TaqMan assay had equal or higher sensitivity and was able to discriminate correctly the viral species in all tested samples, whereas a real-time RT-PCR assay previously developed for HoBi-like pestivirus detection showed cross-reactivity with few high-titre BVDV-2 samples.
authors.affiliation: Department of Veterinary Medicine, University of Bari, Strada per Casamassima Km 3, 70010 Valenzano (Bari), Italy.
authors.forename: Viviana
authors.initials: V
authors.lastname: Mari
authors.affiliation: Department of Veterinary Medicine, University of Bari, Strada per Casamassima Km 3, 70010 Valenzano (Bari), Italy.
authors.forename: Michele
authors.initials: M
authors.lastname: Losurdo
authors.affiliation: Department of Veterinary Medicine, University of Bari, Strada per Casamassima Km 3, 70010 Valenzano (Bari), Italy.
authors.forename: Maria Stella
authors.initials: MS
authors.lastname: Lucente
authors.affiliation: Department of Veterinary Medicine, University of Bari, Strada per Casamassima Km 3, 70010 Valenzano (Bari), Italy.
authors.forename: Eleonora
authors.initials: E
authors.lastname: Lorusso
authors.affiliation: Department of Veterinary Medicine, University of Bari, Strada per Casamassima Km 3, 70010 Valenzano (Bari), Italy; Specialization School in Infectious Diseases of Animals, University of Bari, Strada per Casamassima Km 3, 70010 Valenzano (Bari), Italy.
authors.forename: Gabriella
authors.initials: G
authors.lastname: Elia
authors.affiliation: Department of Veterinary Medicine, University of Bari, Strada per Casamassima Km 3, 70010 Valenzano (Bari), Italy; Specialization School in Infectious Diseases of Animals, University of Bari, Strada per Casamassima Km 3, 70010 Valenzano (Bari), Italy.
authors.forename: Vito
authors.initials: V
authors.lastname: Martella
authors.affiliation: Specialization School in Infectious Diseases of Animals, University of Bari, Strada per Casamassima Km 3, 70010 Valenzano (Bari), Italy.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Patruno
authors.affiliation: Department of Veterinary Medicine, University of Bari, Strada per Casamassima Km 3, 70010 Valenzano (Bari), Italy; Specialization School in Infectious Diseases of Animals, University of Bari, Strada per Casamassima Km 3, 70010 Valenzano (Bari), Italy.
authors.forename: Domenico
authors.initials: D
authors.lastname: Buonavoglia
authors.affiliation: Department of Veterinary Medicine, University of Bari, Strada per Casamassima Km 3, 70010 Valenzano (Bari), Italy; Specialization School in Infectious Diseases of Animals, University of Bari, Strada per Casamassima Km 3, 70010 Valenzano (Bari), Italy. Electronic address: nicola.decaro@uniba.it.
authors.forename: Nicola
authors.initials: N
authors.lastname: Decaro
mesh_terms: D000818:Animals; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis* / Q000821:virology; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D010576:Pestivirus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D018182:Pestivirus Infections / Q000662:veterinary* / Q000821:virology; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
keywords: Cattle; Discrimination; HoBi-like pestivirus; Multiplex real-time RT-PCR; Pestivirus