Department of Plant Pathology, Washington State University, Prosser, WA 99350, United States.
Department of Plant Pathology, Washington State University, Prosser, WA 99350, United States.
J Virol Methods. 2020 Apr;278:113836. doi: 10.1016/j.jviromet.2020.113836. Epub 2020 Feb 19.
Latent fruit tree viruses present economic threat to the industry and nurseries as diseases they cause not only reduce fruit quality and production yield, but can also be spread inadvertently through propagation due to the lack of viral symptoms on an infected mother plant. As a result, these viruses require appropriate detection tools for effective management. In this study we developed RT-qPCR assays for the detection of three latent viruses of pome, apple chlorotic leaf spot virus (ACLSV), apple stem pitting virus (ASPV), and apple mosaic virus (ApMV), using the alignment of representative sequences from the NCBI database. The optimized assays were shown to be specific by successfully amplifying the target from positive controls without showing any detectable amplification in negative and non-target controls, and revealed high sensitivity by reliably detecting as low as 10 copies per reaction. The results also demonstrated that both the choice of extraction method and the reagents used for RT-qPCRcould play a critical role in virus detection outcome. These assays were both reliable and robust compared to the extant RT-PCR methods, and they could be a viable tool for making informed management decisions.
潜伏果树病毒对行业和苗圃构成经济威胁，因为它们引起的疾病不仅会降低果实质量和产量，而且由于受感染母株上缺乏病毒症状，还可能在无意中通过繁殖传播。因此，这些病毒需要适当的检测工具才能进行有效管理。在这项研究中，我们使用来自 NCBI 数据库的代表性序列进行比对，为检测三种潜伏性苹果病毒（李属坏死环斑病毒（ACLSV）、苹果茎陷点病毒（ASPV）和苹果花叶病毒（ApMV））开发了 RT-qPCR 检测方法。优化后的检测方法通过成功地从阳性对照中扩增目标，而在阴性对照和非目标对照中没有显示出任何可检测的扩增，证明了其特异性，并且通过可靠地检测低至每个反应 10 个拷贝的病毒，显示出了很高的灵敏度。结果还表明，提取方法和 RT-qPCR 所用试剂的选择对病毒检测结果起着至关重要的作用。与现有的 RT-PCR 方法相比，这些检测方法既可靠又稳健，它们可能成为做出明智管理决策的可行工具。
