title: A multiplex DNA suspension microarray for simultaneous detection and differentiation of classical swine fever virus and other pestiviruses.
abstract: An oligonucleotide suspension microarray (Luminex microsphere system) was developed for detection and differentiation of animal pestiviruses: classical swine fever virus (CSFV), bovine viral diarrhea virus types 1 and 2 (BVDV1 and BVDV2), and border disease virus (BDV). Species-specific and pestivirus-common oligonucleotide probes were designed to the 5' UTR region and conjugated to individual color-coded Luminex carboxy beads (probe beads). Target pestivirus sequences were amplified by asymmetric PCR using a biotinylated reverse primer and a forward and reverse primer ratio of 1:5. The biotinylated products were hybridized to eight probe beads in a multiplex assay and analyzed using streptavidin conjugated to a fluorescent reporter molecule. The assay was able to detect and differentiate all 40 strains of CSFV, BVDV1, BVDV2 and BDV tested. The analytical sensitivity was determined to be 0.2-10 TCID50/ml. The major advantages of the DNA-microsphere suspension microarray, as a low density array, are its ease of handling and ability to simultaneously detect and type multiple infectious agents.
authors.affiliation: Virology Section, Lethbridge Laboratory, Animal Diseases Research Institute, Canadian Food Inspection Agency, Lethbridge, Alta., Canada T1J 3Z4. deregtd@inspection.gc.ca
authors.forename: Dirk
authors.initials: D
authors.lastname: Deregt
authors.forename: Scott A
authors.initials: SA
authors.lastname: Gilbert
authors.forename: Sandor
authors.initials: S
authors.lastname: Dudas
authors.forename: John
authors.initials: J
authors.lastname: Pasick
authors.forename: Shailja
authors.initials: S
authors.lastname: Baxi
authors.forename: Kimberley M
authors.initials: KM
authors.lastname: Burton
authors.forename: Mohit K
authors.initials: MK
authors.lastname: Baxi
mesh_terms: D020121:5' Untranslated Regions / Q000235:genetics; D001710:Biotin; D005453:Fluorescence; D005838:Genotype; D008863:Microspheres; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D010576:Pestivirus / Q000145:classification* / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D012367:RNA, Viral / Q000235:genetics; D012680:Sensitivity and Specificity; D013194:Staining and Labeling; D019809:Streptavidin
chemical_list: D020121:5' Untranslated Regions; D012367:RNA, Viral; D001710:Biotin; D019809:Streptavidin