Colorimetric diagnosis of prolonged bluetongue viremia in sheep, using an enzyme-linked oligonucleotide sorbent assay of amplified viral nucleic acids

Abstract

Summary Each of 5 US-origin serotypes of bluetongue virus ( btv ) was inoculated into a separate pair of sheep. The duration of each animal's ensuing viremia was monitored, using a btv serogroup-specific nested polymerase chain reaction ( pcr ) method and an embryonating chicken egg ( ece ) inoculation procedure. Mean duration of viremia was 100 and 38 days for the pcr and ece methods, respectively. This difference was significant ( P < 0.001) and documents a more prolonged viremia in virus-exposed sheep than has been reported. A dual internal oligonucleotide solution hybridization procedure was developed for the rapid (2 hours) colorimetric detection and identification of btv -specific pcr products. This enzyme-linked oligonucleotide sorbent assay ( elosa ) relied on annealing of separate biotinylated and fluores-ceinated probes to the amplified btv nucleic acid; these complexes were captured on streptavidin-coated microtitration wells and were detected, using a horseradish peroxidase-labeled antifluorescein antibody conjugate. End-point dilution analyses of pcr products indicated that the elosa was more sensitive than gel electrophoretic or comparable colorimetric slot-blot hybridization techniques. The btv pcr - elosa system represents a more sensitive and expeditious means of diagnosing btv -induced viremia than does the ece procedure currently used. The combination of elosa with pcr should facilitate practical application of nucleic acid technology to diagnostic veterinary medicine.