Journal of Virological Methods 179 (2012) 419–422 Contents lists available at SciVerse ScienceDirect Journal of Virological Methods jou rn al h om epage: www.elsevier.com/locate/jviromet Short communication Validation of a high-throughput real-time polymerase chain reaction assay for the detection of capripoxviral DNA Samuel Stubbs a,b , Chris A.L. Oura a , Mark Henstock a , Timothy R. Bowden c , Donald P. King a , Eeva S.M. Tuppurainen a,∗ a Institute for Animal Health, Ash Road, Pirbright, Surrey GU24 0NF, United Kingdom b The Royal Veterinary College, Royal College Street, London NW1 0TU, United Kingdom c CSIRO Livestock Industries, Australian Animal Health Laboratory, Private Bag 24, Geelong, Victoria 3220, Australia Article history: Received 5 April 2011 Received in revised form 30 August 2011 Accepted 16 November 2011 Available online 25 November 2011 Keywords: Capripoxvirus Real-time PCR Diagnosis Validation a b s t r a c t Capripoxviruses, which are endemic in much of Africa and Asia, are the aetiological agents of economically devastating poxviral diseases in cattle, sheep and goats. The aim of this study was to validate a high- throughput real-time PCR assay for routine diagnostic use in a capripoxvirus reference laboratory. The performance of two previously published real-time PCR methods were compared using commercially available reagents including the amplification kits recommended in the original publication. Furthermore, both manual and robotic extraction methods used to prepare template nucleic acid were evaluated using samples collected from experimentally infected animals. The optimised assay had an analytical sensitivity of at least 63 target DNA copies per reaction, displayed a greater diagnostic sensitivity compared to conventional gel-based PCR, detected capripoxviruses isolated from outbreaks around the world and did not amplify DNA from related viruses in the genera Orthopoxvirus or Parapoxvirus. The high-throughput robotic DNA extraction procedure did not adversely affect the sensitivity of the assay compared to manual preparation of PCR templates. This laboratory-based assay provides a rapid and robust method to detect capripoxviruses following suspicion of disease in endemic or disease-free countries. Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved. Lumpy skin disease (LSD), sheeppox (SPP) and goatpox (GTP) are economically important viral diseases of domestic ruminants, primarily affecting cattle, sheep and goats, respectively (Tulman et al., 2002). In a susceptible population the morbidity rate associ- ated with SPP and GTP can reach 100%, and the mortality rates can approach 50% in adults and 100% in young stock (Bhanuprakash et al., 2006). In contrast, LSD outbreaks generally result in low mortality (1–3%) and variable morbidity, averaging 10%, although rates as high as 85% are not uncommon (Babiuk et al., 2008a,b; Bowden et al., 2009). The aetiological agents of these diseases are lumpy skin disease virus (LSDV), sheeppox virus (SPPV) and goat- pox virus (GTPV). They are large, complex, double-stranded DNA viruses of the genus Capripoxvirus, subfamily Chordopoxvirinae, family Poxviridae (Buller et al., 2005). Capripoxviruses are endemic in much of the developing world where they cause significant eco- nomic losses due to decreased milk yield, weight loss, infertility in males and females, and damage to hides and wool (Yeruham et al., 2007). The occurrence of LSD is restricted to Africa and the Middle East, whereas the geographical distribution of SPP and GTP over- laps with that of LSD in equatorial Africa, from where they extend ∗ Corresponding author. Tel.: +44 01483 231149; fax: +44 01483 235745. E-mail address: eeva.tuppurainen@iah.ac.uk (E.S.M. Tuppurainen). to Northern Africa and Asia, including the Middle East (Babiuk et al., 2008a,b). Rapid and accurate laboratory tests for the detection of SPPV, GTPV and LSDV are essential for confirming the diagnosis of disease following outbreaks in either endemic or disease-free coun- tries. These assays enable appropriate control measures to be implemented to limit the spread and prevent establishment of dis- ease. Both conventional and real-time polymerase chain reaction (PCR) assays for detection of capripoxviruses have been reported, with better sensitivity and specificity than antigen-trapping ELISA (Carn and Kitching, 1995) or virus isolation (Balinsky et al., 2008; Bowden et al., 2008; Heine et al., 1999; Ireland and Binepal, 1998; Markoulatos et al., 2000; Tuppurainen et al., 2005; Zheng et al., 2007). Furthermore, conventional and real-time PCR methods that enable the differentiation of SPPV, GTPV and LSDV have also been described (Lamien et al., 2011; Orlova et al., 2006). Real-time PCR assays are quantitative, simpler and faster to perform compared to conventional PCR, and are therefore better suited to high- throughput testing of samples. A number of real-time PCR methods for the detection of capripoxviruses have recently been described (Balinsky et al., 2008; Bhanuprakash et al., 2008; Bowden et al., 2008). Validation of the assay designed by Balinsky et al. (2008) was undertaken using swabs, buffy coats and skin lesions collected from 8 lambs experimentally infected with SPPV, as well as various 0166-0934/$ – see front matter. Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2011.11.015