title: Detection of bovine coronavirus using a TaqMan-based real-time RT-PCR assay.
abstract: A real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for the detection of bovine coronavirus (BCoV) RNA in clinical samples is described. The assay is based on TaqMan technology, consisting of two primers and one probe labeled with the reporter dye 6-carboxyfluorescein that binds selectively to the transmembrane-protein gene of BCoV. The BCoV real-time RT-PCR assay was able to detect the tested BCoV and BCoV-like viruses (canine respiratory coronavirus and bubaline coronavirus), whereas other common viral pathogens of cattle were not recognised by the established oligonucleotide set, thus showing that the test was specific for bovine-like CoVs. The detection limit of the assay was 20 BCoV RNA copies (1-log higher with respect to traditional gel-based RT-PCR) and the reproducibility was satisfactory, thus allowing for a sensitive and accurate measurement of the viral RNA load in clinical samples. Two hundred and twenty clinical specimens (92 rectal, 82 nasal and 46 ocular swabs) were subjected to gel-based and real-time RT-PCR. By conventional amplification, 43 rectal, 54 nasal and 34 ocular samples tested positive, whereas the TaqMan assay was able to detect the BCoV nucleic acid in 49 rectal, 60 nasal and 37 ocular swabs. The rapidity and high throughput of the BCoV TaqMan assay makes this method a powerful tool for a sensitive and specific diagnosis of BCoV infection in cattle.
authors.affiliation: Department of Public Health and Animal Sciences, Faculty of Veterinary Medicine of Bari, Strada per Casamassima Km 3, 70010 Valenzano, Bari, Italy.
authors.forename: Nicola
authors.initials: N
authors.lastname: Decaro
authors.forename: Gabriella
authors.initials: G
authors.lastname: Elia
authors.forename: Marco
authors.initials: M
authors.lastname: Campolo
authors.forename: Costantina
authors.initials: C
authors.lastname: Desario
authors.forename: Viviana
authors.initials: V
authors.lastname: Mari
authors.forename: Arianna
authors.initials: A
authors.lastname: Radogna
authors.forename: Maria Loredana
authors.initials: ML
authors.lastname: Colaianni
authors.forename: Francesco
authors.initials: F
authors.lastname: Cirone
authors.forename: Maria
authors.initials: M
authors.lastname: Tempesta
authors.forename: Canio
authors.initials: C
authors.lastname: Buonavoglia
mesh_terms: D000818:Animals; D002417:Cattle; D002418:Cattle Diseases / Q000821:virology; D018352:Coronavirus Infections / Q000175:diagnosis / Q000662:veterinary*; D017938:Coronavirus, Bovine / Q000235:genetics* / Q000302:isolation & purification*; D005123:Eye / Q000821:virology; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods
chemical_list: D012367:RNA, Viral