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.

An appraisal of different methods for the detection of the walnut strain of cherry leafroll virus

“…These reports describe methods that use a previous step for RNA purification. Extraction of undamaged RNA for specific priming of an efficient RT reaction to obtain proper templates for amplification has been described as a major problem (Verbeek and Tijssen, 1990;Chirnside et al, 1990). We have not found it to be necessary in order to obtain high sensitivity for wCLRV detection in tobacco or walnut.…”

Multiplex semi-nested RT-PCR with exogenous internal control for simultaneous detection of bovine coronavirus and group A rotavirus

“…Detection of BCoV has been carried out using ELISA with monoclonal and polyclonal antibodies, immunohistochemistry assays (Clark, 1993;Zhang et al, 1997;Schoenthaler and Kapil, 1999), the polymerase chain reaction (PCR) (Verbeek and Tijssen, 1990;Loa et al, 2006;Takiuchi et al, 2006), and real-time PCR (Escutenaire et al, 2007;Decaro et al, 2008;Cho et al, 2010).…”

Genomic relationship between turkey and bovine enteric coronaviruses identified by hybridization with BCV or TCV specific cDNA probes

“…Several recombinant plasmids, containing BCVspecific cDNA sequences [33,34], were radioactively labelled by nick translation and used None or in combination for the detection of TCV. Polymerase chain reaction (PCR)-produced probes which were highly efficient in detection of BCV [35], were also used to detect purified TCV or virus in supernatants of infected cell cultures. The use of one recombinant plasmid, either BCV or TCV specific, was usually unsufficient for the detection of TCV in clinical samples from diarrheic pours.…”