Journal
JOURNAL OF VIROLOGICAL METHODS
Volume 136, Issue 1-2, Pages 17-23Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jviromet.2006.03.025
Keywords
DNA suspension microarray; pestivirus; classical swine fever virus
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An oligonucleotide suspension microarray (Luminex((R)) microsphere system) was developed for detection and differentiation of animal pestiviruses: classical swine fever virus (CSFV), bovine viral diarrhea virus types I and 2 (BVDV I 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 0.2-10TCID(50)/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. Crown Copyright (c) 2006 Published by Elsevier B.V. All rights reserved.
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