title: Multiplex RT-PCR and Automated Microarray for Detection of Eight Bovine Viruses.
abstract: Microarrays can be a useful tool for pathogen detection as it allow for simultaneous interrogation of the presence of a large number of genetic sequences in a sample. However, conventional microarrays require extensive manual handling and multiple pieces of equipment for printing probes, hybridization, washing and signal detection. In this study, a reverse transcription (RT)-PCR with an accompanying novel automated microarray for simultaneous detection of eight viruses that affect cattle [vesicular stomatitis virus (VSV), bovine viral diarrhoea virus type 1 and type 2, bovine herpesvirus 1, bluetongue virus, malignant catarrhal fever virus, rinderpest virus (RPV) and parapox viruses] is described. The assay accurately identified a panel of 37 strains of the target viruses and identified a mixed infection. No non-specific reactions were observed with a panel of 23 non-target viruses associated with livestock. Vesicular stomatitis virus was detected as early as 2 days post-inoculation in oral swabs from experimentally infected animals. The limit of detection of the microarray assay was as low as 1 TCID50/ml for RPV. The novel microarray platform automates the entire post-PCR steps of the assay and integrates electrophoretic-driven capture probe printing in a single user-friendly instrument that allows array layout and assay configuration to be user-customized on-site.
authors.affiliation: Lethbridge Laboratory, National Centres for Animal Disease, Canadian Food Inspection Agency, Lethbridge, AB, Canada.
authors.forename: O
authors.initials: O
authors.lastname: Lung
authors.affiliation: Lethbridge Laboratory, National Centres for Animal Disease, Canadian Food Inspection Agency, Lethbridge, AB, Canada.
authors.forename: T
authors.initials: T
authors.lastname: Furukawa-Stoffer
authors.affiliation: Lethbridge Laboratory, National Centres for Animal Disease, Canadian Food Inspection Agency, Lethbridge, AB, Canada.
authors.forename: K
authors.initials: K
authors.lastname: Burton Hughes
authors.affiliation: National Centre for Foreign Animal Disease, Canadian Food Inspection Agency, Winnipeg, MB, Canada.
authors.forename: J
authors.initials: J
authors.lastname: Pasick
authors.affiliation: The Pirbright Institute, Woking, Surrey, UK.
authors.forename: D P
authors.initials: DP
authors.lastname: King
authors.affiliation: Nexogen, Inc., San Diego, CA, USA.
authors.forename: D
authors.initials: D
authors.lastname: Hodko
mesh_terms: D000818:Animals; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis* / Q000821:virology; D060085:Coinfection / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods / Q000662:veterinary*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012680:Sensitivity and Specificity; D014777:Virus Diseases / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D014780:Viruses / Q000302:isolation & purification
keywords: automation; bluetongue; bovine herpesvirus; bovine viral diarrhoea; malignant catarrhal fever; microarray; multiplex PCR; rinderpest