A multiplex DNA suspension microarray for simultaneous detection and differentiation of classical swine fever virus and other pestiviruses.
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.
Deregt
Dirk
D
Virology Section, Lethbridge Laboratory, Animal Diseases Research Institute, Canadian Food Inspection Agency, Lethbridge, Alta., Canada T1J 3Z4. deregtd@inspection.gc.ca
Gilbert
Scott A
SA
Dudas
Sandor
S
Pasick
John
J
Baxi
Shailja
S
Burton
Kimberley M
KM
Baxi
Mohit K
MK
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
D020121:5' Untranslated Regions; D012367:RNA, Viral; D001710:Biotin; D019809:Streptavidin