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

Verbeek, Arnold; Tijssen, Peter

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 × 105 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 × 104 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.