TaqMan based real-time duplex PCR for simultaneous detection and quantitation of capripox and orf virus genomes in clinical samples

A rapid and sensitive TaqMan based real-time duplex PCR (drt-PCR) assay for simultaneous detection, differentiation and quantitation of Capripoxvirus (CaPV) and Orf virus (ORFV) DNA, was optimized targeting the highly conserved DNA polymerase genes of these virus genomes. Two pairs of oligonucleotide primers and two hybridization probes labeled with Cy5/BHQ1 and Hex/BHQ1 for CaPV and ORFV, respectively, were used in the drt-PCR assay. The assay was found to be specific only to targeted viruses and did not react with buffalopox virus (BPXV), camelpox virus (CMLV) (Orthopoxviruses) and cDNA of Peste des petits ruminants virus and bluetongue virus, the other common viruses of sheep and goats. The detection limit of the assay was 20 copies for each of the standard plasmid and 35 fg of viral genomic DNA for CaPV and ORFV, respectively, in a single and mixed virus population. Both intra-(0.49-4.6% and 0.7-3.7%) and inter-(0.6-2.35% and 0.27-2.1%) assay variations of drt-PCR for CaPV and ORFV DNA were within the acceptable limits, implying high reproducibility and repeatability of the assay. Further, the diagnostic specificity and the sensitivity of the assay was assessed using known virus isolates of sheeppox virus (SPPV), goatpox virus (GTPV) and ORFV and the clinical specimens from sheep and goats. The developed drt-PCR assay was able to detect, differentiate, quantify simultaneously and also to identity mixed infections of CaPV and ORFV in sheep and goats.