Istituto di Virologia Vegetale del CNR, UOS Bari, Via Amendola 165/A, 70126 Bari, Italy.
J Virol Methods. 2012 Dec;186(1-2):141-6. doi: 10.1016/j.jviromet.2012.08.005. Epub 2012 Aug 28.
The spread of viroids belonging to the genus Pospiviroid (family Pospiviroidae), recorded recently in ornamentals and vegetables in several European countries, calls for fast, efficient and sensitive detection methods. Based on bioinformatics analyses of sequence identity among all pospiviroids, a digoxigenin-labeled polyprobe (POSPIprobe) was developed that, when tested by dot-blot and Northern-blot hybridization, detected Potato spindle tuber viroid, Citrus exocortis viroid, Columnea latent viroid, Mexican papita viroid, Tomato planta macho viroid, Tomato apical stunt viroid, Pepper chat fruit viroid and Chrysanthemum stunt viroid. The end-point detection limits of the POSPIprobe ranged from 5(-2) to 5(-4), and from 5(-1) to 5(-3) for nucleic acid preparations obtained by phenol extraction and silica-capture, respectively, similar to those of single probes. Based on sequence identity, the POSPIprobe is expected to detect also the two pospiviroid species not tested in this study (Tomato chlorotic dwarf viroid and Iresine viroid-1). Dot-blot assays with the POSPIprobe were validated by testing 68 samples from tomato, chrysanthemum and argyranthemum infected by different pospiviroids as revealed by RT-PCR, thus confirming the potential of this polyprobe for quarantine, certification and survey programs.
最近在几个欧洲国家的观赏植物和蔬菜中记录到属于 Pospiviroid 属的类病毒的传播，这需要快速、高效和敏感的检测方法。基于对所有 Pospiviroids 序列同一性的生物信息学分析，开发了一种地高辛标记的多探针 (POSPIprobe)，通过点印迹和 Northern 印迹杂交试验，该探针可检测马铃薯纺锤块茎类病毒、柑橘外果皮类病毒、Columnea 潜伏类病毒、墨西哥南瓜类病毒、番茄植株雄性病毒、番茄顶端生长迟缓病毒、辣椒聊天果类病毒和菊花矮缩类病毒。POSPIprobe 的终点检测限范围为 5(-2) 至 5(-4)，对于用苯酚提取和硅捕获获得的核酸制剂，分别为 5(-1) 至 5(-3)，与单探针的检测限相似。基于序列同一性，预计 POSPIprobe 还可以检测本研究中未测试的两种 Pospiviroid 物种（番茄黄花矮缩病毒和 Iresine 类病毒-1）。通过用 RT-PCR 检测经不同 Pospiviroids 感染的番茄、菊花和大丁草的 68 个样本进行 dot-blot 分析，验证了 POSPIprobe 的有效性，从而证实了这种多探针在检疫、认证和调查计划中的潜力。
