The Pirbright Institute, Pirbright, Surrey, UK.
BMC Vet Res. 2013 May 1;9:90. doi: 10.1186/1746-6148-9-90.
Sheep poxvirus (SPPV), Goat poxvirus (GTPV) and Lumpy skin disease virus (LSDV) are the most serious poxviruses of ruminants. They are double stranded DNA viruses of the genus Capripoxvirus, (subfamily Chordopoxvirinae) within the family Poxviridae. The aim of this study was to develop a Loop-mediated isothermal AMPlification (LAMP) assay for the detection of Capripoxvirus (CaPV) DNA.
A single LAMP assay targeting a conserved region of the CaPV P32 gene was selected from 3 pilot LAMP assays and optimised by adding loop primers to accelerate the reaction time. This LAMP assay successfully detected DNA prepared from representative CaPV isolates (SPPV, GTPV and LSDV), and did not cross-react with DNA extracted from other mammalian poxviruses. The analytical sensitivity of the LAMP assay was determined to be at least 163 DNA copies/μl which is equivalent to the performance reported for diagnostic real-time PCR currently used for the detection of CaPV. LAMP reactions were monitored with an intercalating dye using a real-time PCR machine, or by agarose-gel electrophoresis. Furthermore, dual labelled LAMP products (generated using internal LAMP primers that were conjugated with either biotin or fluorescein) could be readily visualised using a lateral-flow device.
This study provides a simple and rapid approach to detect CaPV DNA that may have utility for use in the field, or in non-specialised laboratories where expensive equipment is not available.
绵羊痘病毒（SPPV）、山羊痘病毒（GTPV）和牛结节疹病毒（LSDV）是反刍动物中最严重的痘病毒。它们是双股 DNA 病毒，属于痘病毒科（Chordopoxvirinae 亚科）的 Capripoxvirus 属。本研究旨在开发一种用于检测 Capripoxvirus（CaPV）DNA 的环介导等温扩增（LAMP）检测方法。
从 3 个初步的 LAMP 试验中选择了一个针对 CaPV P32 基因保守区域的单一 LAMP 试验，并通过添加环引物来优化反应时间。该 LAMP 试验成功地检测到了来自代表性 CaPV 分离株（SPPV、GTPV 和 LSDV）的 DNA，并且与从其他哺乳动物痘病毒提取的 DNA 没有交叉反应。LAMP 试验的分析灵敏度至少为 163 个 DNA 拷贝/μl，与目前用于检测 CaPV 的诊断实时 PCR 的性能相当。可以使用实时 PCR 仪或琼脂糖凝胶电泳来监测 LAMP 反应。此外，使用内部 LAMP 引物（与生物素或荧光素缀合）生成的双标记 LAMP 产物可以使用侧流装置轻松可视化。
本研究提供了一种简单快速的检测 CaPV DNA 的方法，可能适用于现场或没有昂贵设备的非专业实验室。
