title: Polymerase chain reaction for probe synthesis and for direct amplification in detection of bovine coronavirus.
abstract: The polymerase chain reaction (PCR) was used to synthesize ds and ss probes for the detection of bovine coronavirus (BCV) using recombinant plasmids as template molecules. The ds probes detected a minimum of about 2 X 10(5) viral genomes after exposure for 1 h, a detection limit similar to nick-translated probes after exposure of the films for 60 h. More than 8 h exposure to blots probed with these ds probes resulted in complete darkening of the film. The ss probes, synthesized by asymmetric PCR on linearized plasmids, permitted the detection of 5 X 10(4) genomes, which equalled the capacity of random-primed probes. Prolonged exposure did not increase the background as in case of ds PCR-probed blots. Probes, synthesized by asymmetric PCR and random-priming were labeled to similar specific activities and were better in terms of sensitivity and detectability as opposed to nick-translated probes. However, the specificity of detection with ss probes as to random primed probes was increased further. About 10 viral genomes, after fragment-specific amplification by PCR, were detected by agarose-gel analysis. PCR-probe synthesis was simple, highly reproducible, and allowed the synthesis of probes for specific fragments.
authors: Centre de recherche en médicine comparée, Institut Armand-Frappier, Université du Québec, Laval-des-Rapides, Canada.
authors: A
authors: A
authors: Verbeek
authors: P
authors: P
authors: Tijssen
mesh_terms: D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003332:Coronaviridae / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D009841:Oligonucleotides; D010957:Plasmids; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D015347:RNA Probes; D012367:RNA, Viral / Q000032:analysis
chemical_list: D009841:Oligonucleotides; D015347:RNA Probes; D012367:RNA, Viral