Department of Plant Pathology, University of Wisconsin-Madison, 1630 Linden Drive, Madison, WI, 53706, USA.
Research Center for Global Agromedicine, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Inada-cho, Obihiro, 0808555, Japan.
J Virol Methods. 2019 May;267:16-21. doi: 10.1016/j.jviromet.2019.02.006. Epub 2019 Feb 20.
Potato virus Y (PVY) is a global challenge for potato production and the leading cause of seed crop downgrading and rejection for certification. Accurate and timely diagnosis is key to effective control of PVY. Here we optimized the isothermal recombinase polymerase amplification (RPA) for accurate detection of different PVY O and N types that were tested, present in different tissues of potato plants including tubers with a primer set that specifically targets the highly conserved pipo region within the viral genome. Combined with a simplified preparation of the template by tissue homogenization, we established a rapid RPA procedure, which can allow real time detection in less than 10 min with a fluorescent probe. Specificity of the reaction was determined by the lack of cross-reactivity with other common potato viruses. Although RPA reagents remain more expensive than PCR reagents, RPA technology is equivalent in that results can be visualized by gel electrophoresis or with a fluorescent probe with greater sensitivity; and it is superior to the common PCR-based assay in its versatility, speed, and lack of need for a highly purified RNA template.
马铃薯 Y 病毒（PVY）是马铃薯生产面临的全球性挑战，也是导致种薯降级和认证拒收的主要原因。准确、及时的诊断是有效控制 PVY 的关键。在这里，我们优化了等温重组酶聚合酶扩增（RPA）技术，以准确检测不同的 PVY O 和 N 型，这些病毒存在于马铃薯植株的不同组织中，包括块茎，使用的引物组针对病毒基因组中高度保守的 pipo 区域。该方法与组织匀浆简化模板制备相结合，建立了一种快速 RPA 程序，通过荧光探针可在不到 10 分钟的时间内进行实时检测。该反应的特异性是通过缺乏与其他常见马铃薯病毒的交叉反应来确定的。虽然 RPA 试剂仍然比 PCR 试剂昂贵，但 RPA 技术在结果可视化方面与 PCR 相当，可通过凝胶电泳或荧光探针进行检测，具有更高的灵敏度；而且与常见的基于 PCR 的检测方法相比，RPA 技术具有通用性、速度快和不需要高度纯化的 RNA 模板等优势。
