title: Validation of a high-throughput real-time polymerase chain reaction assay for the detection of capripoxviral DNA.
abstract: 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.
authors.affiliation: Institute for Animal Health, Pirbright, Surrey GU24 0NF, United Kingdom.
authors.forename: Samuel
authors.initials: S
authors.lastname: Stubbs
authors.forename: Chris A L
authors.initials: CA
authors.lastname: Oura
authors.forename: Mark
authors.initials: M
authors.lastname: Henstock
authors.forename: Timothy R
authors.initials: TR
authors.lastname: Bowden
authors.forename: Donald P
authors.initials: DP
authors.lastname: King
authors.forename: Eeva S M
authors.initials: ES
authors.lastname: Tuppurainen
mesh_terms: D000818:Animals; D001331:Automation / Q000379:methods; D018151:Capripoxvirus / Q000235:genetics / Q000302:isolation & purification*; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis / Q000821:virology; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D015511:Goat Diseases / Q000175:diagnosis / Q000821:virology; D006041:Goats; D057166:High-Throughput Screening Assays / Q000379:methods*; D011213:Poxviridae Infections / Q000175:diagnosis / Q000662:veterinary*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012756:Sheep; D012757:Sheep Diseases / Q000175:diagnosis / Q000821:virology; D014730:Veterinary Medicine / Q000379:methods*; D014773:Virology / Q000379:methods*
chemical_list: D004279:DNA, Viral