Plant Health and Environment Laboratory, Investigation and Diagnostic Centre, MAF Biosecurity New Zealand, Auckland, New Zealand.
J Virol Methods. 2011 Jan;171(1):190-4. doi: 10.1016/j.jviromet.2010.10.023. Epub 2010 Oct 27.
A TaqMan-based real-time one-step RT-PCR assay was developed for the rapid detection of Tomato black ring virus (TBRV), a significant plant pathogen which infects a wide range of economically important crops. Primers and a probe were designed against existing genomic sequences to amplify a 72 bp fragment from RNA-2. The assay amplified all isolates of TBRV tested, but no amplification was observed from the RNA of other nepovirus species or healthy host plants. The detection limit of the assay was estimated to be around nine copies of the TBRV target region in total RNA. A comparison with conventional RT-PCR and ELISA, indicated that ELISA, the current standard test method, lacked specificity and reacted to all nepovirus species tested, while conventional RT-PCR was approximately ten-fold less sensitive than the real-time RT-PCR assay. Finally, the real-time RT-PCR assay was tested using five different RT-PCR reagent kits and was found to be robust and reliable, with no significant differences in sensitivity being found. The development of this rapid assay should aid in quarantine and post-border surveys for regulatory agencies.
建立了一种基于 TaqMan 的实时一步 RT-PCR 检测方法，用于快速检测番茄黑环病毒（TBRV）。TBRV 是一种重要的植物病原体，可感染广泛的经济重要作物。引物和探针是根据现有基因组序列设计的，用于从 RNA-2 扩增 72bp 片段。该检测方法可扩增所有测试的 TBRV 分离株，但未从其他 nepovirus 物种的 RNA 或健康宿主植物中观察到扩增。该检测方法的检测限估计约为总 RNA 中 TBRV 靶区的 9 个拷贝。与常规 RT-PCR 和 ELISA 的比较表明，ELISA（当前的标准测试方法）缺乏特异性，并且与所有测试的 nepovirus 物种发生反应，而常规 RT-PCR 的灵敏度比实时 RT-PCR 检测方法低约十倍。最后，使用五种不同的 RT-PCR 试剂盒对实时 RT-PCR 检测方法进行了测试，结果表明该方法稳健可靠，灵敏度无显著差异。这种快速检测方法的开发应该有助于检疫和监管机构的边境后调查。
