A real time PCR assay for the detection and quantification of orf virus

A real time quantitative PCR assay based on TaqMan® technology was developed for orf virus (ORFV) DNA quantification in clinical samples, infected cells and organotypic cultures. This method was based on the amplification of a 70 bp fragment from the ORFV B2L gene (orthologue of the Vaccinia virus Copenhagen F13L gene) that encodes the major envelope protein. Both intra- and inter-assay variability were well within ±0.25 log10 S.D. showing the high efficiency and reproducibility of the assay. The TaqMan® PCR was subsequently used to determine the titre of several batches of the ORFV strain NZ-2, with it being possible to quantify virus solutions in the range of 1 × 101 to 1 × 106 TCID50/ml. A good correlation between the titre determined by the TaqMan® PCR and by conventional endpoint dilution was found. The PCR assay is reproducible and can be used for a rapid quantification of ORFV in vitro and ex vivo, being readily achievable within 1 h.