Journal of Virological Methods xxx (2006) xxx–xxx A real time PCR assay for the detection and quantification of orf virus L. Gallina a , F. Dal Pozzo a , C.J. Mc Innes b , G. Cardeti c , A. Guercio d , M. Battilani a , S. Ciulli a , A. Scagliarini a,∗ a Dipartimento di Sanit ` a Pubblica Veterinaria e Patologia Animale, Alma Mater Studiorum, Bologna, via Tolara di Sopra 50, 40064 Ozzano Emilia, Bologna, Italy b Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Midlothian EH26 0PZ, Edinburgh, Scotland c Istituto Zooprofilattico Sperimentale di Lazio e Toscana, via Appia Nuova 1411, 00178 Roma, Italy d Istituto Zooprofilattico Sperimentale della Sicilia via Marinuzzi 3, 90129 Palermo, Italy Received 6 September 2005; received in revised form 12 December 2005; accepted 19 December 2005 Abstract 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 log 10 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 × 10 1 to 1 × 10 6 TCID 50 /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. © 2005 Elsevier B.V. All rights reserved. Keywords: ORFV; Titre determination; Real time PCR; TaqMan ® PCR 1. Introduction Orf virus (ORFV), the prototypic member of the Parapoxviri- dae, is the cause of a papular dermatitis in sheep and goats known as contagious ecthyma (Robinson and Lyttle, 1992). The disease not only has an economic impact on farmers world wide, but also has a considerable effect on animal welfare. Infected animals are sickly, fail to thrive and are more susceptible to adventitious bac- terial infections. Although ORFV vaccines are available these are not used universally as most are fully virulent viruses that can result in outbreaks of disease. Currently there are no effective antiviral treatments available for contagious ecthyma. A conventional PCR assay based on the amplification of part of the ORFV gene (B2L) encoding the major envelope protein (Sullivan et al., 1994) was developed to detect all of the recognised parapoxvirus species and, in conjunction with DNA sequencing, can be utilised to distinguish the different ∗ Corresponding author. Tel.: +39 051 2097083; fax: +39 051 2097039. E-mail address: ascagliarini@vet.unibo.it (A. Scagliarini). species from each other (Inoshima et al., 2000). It has not been used for quantification. Instead, endpoint dilution assays and plaque assays have been used to quantify virus. These assays are time consuming and laborious and generally can not be used for field isolates of ORFV that have not first been adapted for growth in vitro. Real time PCR has proved to be accurate and effective in the quantification of viral DNA and in several virus species a correlation between the quantity of viral DNA and the viral titre has been demonstrated (Gerard et al., 1996; Clark et al., 1999; Espy et al., 2000; Niesters, 2001; Lo and Chao, 2004). As yet no real time PCR has been reported for the parapoxviruses. Here, a real time quantitative PCR is described, based on the amplification of the ORFV B2L gene, that not only could be used for the diagnosis of ORFV associated disease, but also provides a means by which virus titres can be rapidly estimated, poten- tially even from field isolates that do not replicate in vitro. In addition, it is envisaged that the real time PCR assay, in conjunc- tion with raft cultures which mimic ovine skin, could be used to provide a rapid screening method for the efficacy of antiviral drugs. 0166-0934/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2005.12.014 VIRMET-9906; No. of Pages 6