Biotinylated and radioactive cDNA probes in the detection by hybridization of bovine enteric coronavirus.
cDNA, synthesized on bovine coronavirus (BCV) genomic RNA templates, could be used to detect very small quantities (i.e. 1 pg) of viral RNA by hybridization with either radioisotopic-labelled or biotinylated recombinant plasmids. Virus was optimally attached to nitrocellulose membranes when spotted in 1 x SSC, whereas 20 x SSC was superior for viral RNA. Denaturation and RNA fixation of both RNA, still encapsidated in virus particles and isolated genomic RNA, was achieved by baking of the blots in vacuum. Virus detection in the supernatant of infected HRT-18 cells was feasible, but improved significantly after proteinase K treatment. No homology was observed between virus cDNA with either plasmid DNA or nucleic acid isolated from non-infected HRT-18 cells. Hybridization with radioisotopic-labelled probes in higher formamide concentrations (up to 60%) increased the detection signals, possibly by reducing reassociation of the probe. Significant detection amplification (30-50 times) was achieved in the case of biotinylated probes by stimulation of hyperpolymer formation on already hybridized target sequences, by additional hybridization with biotinylated pUC-19. A detection amplification was also obtained when hybridization was done with two probes (pBC-52 and pBC-247), containing non-overlapping viral sequences. Although the detectability was surpassed by biotinylated probes, sensitivity was superior in radioisotopic virus detection.
Verbeek
A
A
Centre de Recherche en Médecine Comparée, Institut Armand-Frappier, Laval-des-Rapides, QC, Canada.
Tijssen
P
P
D000818:Animals; D001710:Biotin; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D003332:Coronaviridae / Q000235:genetics / Q000302:isolation & purification*; D015342:DNA Probes; D005814:Genes, Viral; D009693:Nucleic Acid Hybridization; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification
D015342:DNA Probes; D012367:RNA, Viral; D001710:Biotin