Characterization of nonradioactive hybridization probes for detecting infectious bursal disease virus.
Reverse transcription followed by the polymerase chain reaction was used to amplify a fragment of infectious bursal disease virus (IBDV) strain P3009 genome. The amplified DNA fragment was annealed into the plasmid pUC18 and used to transform Escherichia coli strain JM109. A clone that contained IBDV-specific nucleotide sequences was selected and designated pC23. The DNA fragment within pC23 was 320 base pairs in length and designated C23. Radiolabeled probes prepared from C23 hybridized to genome segment A of strain P3009 by a northern-blot hybridization assay. Biotin-labeled probes prepared from C23 and pC23 either by using nick translation (designated C23/NT and pC23/NT, respectively) or by direct introduction of biotin molecules into C23 and pC32 (designated C23/BH and pC23/BH, respectively) were used in the dot blot hybridization assay for detecting IBDV strains. All four biotinylated probes detected three serotype 1 viruses and one serotype 2 IBDV. However, they did not cross-react with nucleic acids extracted from mock-infected cells or from seven unrelated avian viruses. Probe pC23/BH detected as little as 0.04 ng of IBDV RNA, while the other three probes were less sensitive and detected approximately 1 ng of IBDV RNA. In addition, the probe pC23/BH detected IBDV RNA in bursa tissues from commercial broiler raising farms following the dot blot hybridization.
Lee
L H
LH
Department of Veterinary Medicine, National Chung Hsing University, Taichung, Taiwan, Republic of China.
D000818:Animals; D001483:Base Sequence; D001710:Biotin; D002645:Chickens / Q000382:microbiology; D003001:Cloning, Molecular; D015342:DNA Probes; D004279:DNA, Viral; D007243:Infectious bursal disease virus / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D011201:Poultry Diseases / Q000382:microbiology; D012367:RNA, Viral / Q000235:genetics* / Q000302:isolation & purification; D012088:Reoviridae Infections / Q000382:microbiology / Q000662:veterinary; D012680:Sensitivity and Specificity; D014158:Transcription, Genetic
D015342:DNA Probes; D004279:DNA, Viral; D012367:RNA, Viral; D001710:Biotin