Biotinylated and radioactive cDNA probes in the detection by hybridization of bovine enteric coronavirus

Abstract

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.

Polymerase chain reaction for probe synthesis and for direct amplification in detection of bovine coronavirus

“…Network fo~ation by vector-DNA sequences has been shown to contribute significantly to radioactivity signals (Verbeek and Tijssen, 1988) and is here also evident by comparison of the signals obtained by probing with the isolated cDNAinserts and their corresponding plasmids (Fig. 1, lanes 1 and 2).…”

“…Viral RNA was used for the synthesis of ds cDNA, that was cloned into pUC-9 as previously described (Verbeek and Tijssen, 1988). Recombinant plasmids, used as probes in hybridization assays, were selected for their capacity to hybridize strongly in colony filter hybridization assays.…”

“…Another recombinant plasmid (pN17; Clone N17), containing the coding sequence for the nucleocapsid protein, as verified by sequencing and comparison with a published sequence , was used both as a probe and as a template for probe synthesis by PCR. Recombinant plasmid-(p-52), containing an insert of 660 bp, was previously characterized and used for optimization of hybridization conditions for detection of BCV-RNA (Verbeek and Tijssen, 1988).…”

“…Membranes were baked under vacuum for 90 min at 80°C resulting in virus denaturation and RNA fixation. Blots were then rehydrated in a 6 x SSC solution, prehybridized overnight and hybridized for periods indicated in Results in a solution containing 55% formamide and other components, as previously described (Verbeek and Tijssen, 1988), for probes other than kinase-labeled oligonucleotides. Hybridization with oligonucleotide probes was done at an incubation temperature Ti = T,,,-7, where T,,, was estimated with the formula: T, ("C)= 2(A+T) -C 4(G+C)-7°C (Meinkoth and Wahl, 1984), where G, C, A, T represent the number of each nucleotide contained in the oligonucleotide probe.…”

“…These tests include hemagglutination (Van Balken et al, 1978/79;Sato et al, 1984) and serological methods (Barnett et al, 1975;Crouch et al, 1984;Reynolds et al, 1984;El Ghorr et al, 1988). In the last few years molecular probing of BCV-RNA has been reported for detection either after isolation of the nucleic acid from purified virions (Shockley et al, 1987) or after direct spotting of the virus (Verbeek and Tijssen, 1988). Detection by molecular probing of porcine (Shockley et al, 1987) and human coronaviruses (Myint et al, 1989) in clinical specimens has also been described.…”