National Plant Protection Organization, Wageningen, The Netherlands.
J Virol Methods. 2013 Jan;187(1):43-50. doi: 10.1016/j.jviromet.2012.09.004. Epub 2012 Sep 12.
In many countries phytosanitary regulations apply to Potato spindle tuber viroid, because it can cause serious diseases in potato and tomato crops. Other pospiviroids, some of which are distributed widely in ornamental crops, can cause similar diseases. Consequently, there is a need for a reliable and cost-effective generic testing method. An assay was developed that detects all known species of the genus Pospiviroid, using real-time RT-PCR based on TaqMan technology. This GenPospi assay consists of two reactions running in parallel, the first targeting all pospiviroids, except Columnea latent viroid, the second specifically targeting the latter viroid (already published). To monitor the RNA extraction a nad5 internal control was included. Method validation on tomato leaves showed that the GenPospi assay detects all pospiviroids up to a relative infection rate of 0.13% (equals 770 times dilution). The assay was specific because no cross reactivity was observed with other viroids, viruses or nucleic acid from plant hosts. Repeatability and reproducibility were 100% and the assay appeared robust in an inter-laboratory comparison. The GenPospi assay has been shown to be a suitable tool for large-scale screening for all known pospiviroids. Although it has been validated for tomato leaves it can potentially be used for any crop.
在许多国家，植物卫生法规适用于马铃薯纺锤块茎类病毒，因为它会导致马铃薯和番茄作物的严重疾病。其他 Pospiviroids 也会引起类似的疾病，其中一些广泛分布于观赏作物中。因此，需要一种可靠且具有成本效益的通用测试方法。我们开发了一种使用基于 TaqMan 技术的实时 RT-PCR 检测所有已知 Pospiviroid 属种的检测方法。该 GenPospi 检测法由两个平行运行的反应组成，第一个反应针对除 Columnea 潜伏病毒外的所有 Pospiviroids，第二个反应则特异性地针对后者病毒（已发表）。为了监测 RNA 提取，我们还包括了 nad5 内部对照。在番茄叶片上的方法验证表明，GenPospi 检测法可以检测到所有 Pospiviroids，其相对感染率高达 0.13%（相当于 770 倍稀释）。该检测法具有特异性，因为它没有与其他类病毒、病毒或植物宿主的核酸发生交叉反应。重复性和再现性均为 100%，并且在实验室间比较中该检测法表现出良好的稳健性。GenPospi 检测法已被证明是一种适用于大规模筛查所有已知 Pospiviroids 的工具。虽然它已经在番茄叶片上得到了验证，但它可以潜在地用于任何作物。
