Institute for Animal Health, Pirbright, Surrey GU24 0NF, United Kingdom.
J Virol Methods. 2012 Feb;179(2):419-22. doi: 10.1016/j.jviromet.2011.11.015. Epub 2011 Nov 25.
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.
绵羊痘和山羊痘病毒在非洲和亚洲的大部分地区流行，是导致牛、绵羊和山羊发生具有经济破坏性的痘病毒病的病原体。本研究旨在验证一种高通量实时 PCR 检测方法，以便在绵羊痘病毒参考实验室中常规用于诊断。使用市售试剂比较了两种先前发表的实时 PCR 方法，包括在原始出版物中推荐的扩增试剂盒。此外，还使用从实验感染动物采集的样本评估了用于制备模板核酸的手动和机器人提取方法。优化后的检测方法的分析灵敏度至少为每个反应 63 个靶 DNA 拷贝，与基于凝胶的常规 PCR 相比具有更高的诊断敏感性，可检测到来自世界各地暴发的绵羊痘病毒，并且不会扩增正痘病毒属或副痘病毒属中的相关病毒的 DNA。与手动制备 PCR 模板相比，高通量机器人 DNA 提取程序不会对检测的敏感性产生不利影响。这种基于实验室的检测方法为在流行或无病国家怀疑发生疾病后检测绵羊痘病毒提供了一种快速而可靠的方法。
