title: Improved detection of bovine coronavirus N gene in faeces of calves infected naturally by a semi-nested PCR assay and an internal control.
abstract: Bovine coronavirus (BCoV), a positive sense single-stranded RNA virus, is an important causative agent of neonatal diarrhoea in calves from beef and dairy cattle worldwide. The routine detection and diagnosis of BCoV have been mainly dependent on assays with low sensitivity. The aim of the present study was to develop and evaluate a semi-nested PCR (SN-PCR) to amplify a 251bp fragment of BCoV N gene from fresh (n=25) and frozen (n=25) diarrhoeic faecal samples of naturally infected calves. To improve detection of BCoV in faecal samples by the SN-PCR an internal control was developed, and the results were compared with a conventional RT-PCR assay. The rates of positive samples by SN-PCR and RT-PCR were 24% (12/50) and 8% (4/50), respectively (K=0.43). Only fresh samples were positive in RT-PCR while the SN-PCR detected BCoV in both fresh and frozen faecal samples. The sensitivity of SN-PCR was determined by 10-fold serial dilutions of the BCoV Kakegawa strain (HA titre: 256) that was detected until 10(-7) dilution. The specificity of the amplicons was assessed by restriction fragment length polymorphism and sequence analysis. The inclusion of an internal control provides a way to detect assay inhibition in faecal samples and failure of nucleic acid extraction that allow reduction of the number of false-negative results.
authors.affiliation: Laboratório de Virologia Animal, Departamento de Medicina Veterinária Preventiva, Centro de Ciências Agrárias, Universidade Estadual de Londrina (UEL). Campus Universitário, PO Box 6001, 86051-990, Londrina, Paraná, Brazil.
authors.forename: Elisabete
authors.initials: E
authors.lastname: Takiuchi
authors.forename: Danilo T
authors.initials: DT
authors.lastname: Stipp
authors.forename: Alice F
authors.initials: AF
authors.lastname: Alfieri
authors.forename: Amauri A
authors.initials: AA
authors.lastname: Alfieri
mesh_terms: D000818:Animals; D002417:Cattle; D002418:Cattle Diseases / Q000821:virology*; D018352:Coronavirus Infections / Q000662:veterinary* / Q000821:virology; D000086462:Coronavirus Nucleocapsid Proteins; D017938:Coronavirus, Bovine / Q000235:genetics / Q000302:isolation & purification*; D005243:Feces / Q000821:virology*; D005615:Freezing; D019590:Nucleocapsid Proteins / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012150:Polymorphism, Restriction Fragment Length; D012367:RNA, Viral / Q000032:analysis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013048:Specimen Handling
chemical_list: D000086462:Coronavirus Nucleocapsid Proteins; D019590:Nucleocapsid Proteins; D012367:RNA, Viral