Suzuki Tomo, Ohki Takehiro
Hokkaido Agricultural Research Center, NARO, 1 Hitsujigaoka, Sapporo, Hokkaido 062-8555, Japan; Graduate School of Agriculture, Hokkaido University, N18, W9, Kita-ku, Sapporo, Hokkaido 060-0818, Japan.
J Virol Methods. 2025 Jul;336:115165. doi: 10.1016/j.jviromet.2025.115165. Epub 2025 Apr 10.
Potatoes are susceptible to viral diseases, and widespread viral infections lead to reduced production yields. Potato leaf roll virus, potato virus S, potato virus X, and potato virus Y are important viruses predominant in potato fields and cause significant economic damage in Japan. In this study, to reduce the labor and cost of virus testing in seed certification program, a simple molecular diagnostic assay for these potato viruses was developed. This method consists of template preparation by heat-treating a mixture of leaf homogenate and a newly designed primer set, followed by one-step conventional multiplex RT-PCR (mRT-PCR). In particular, to simplify RNA extraction, a procedure was adopted in which leaf homogenates in PBST were diluted 100-fold in RNase-free water, heat-treated with primers for 5 min at 70°C and subjected to one-step mRT-PCR. Interestingly, heat treatment of a mixture of the homogenate and primers improved the detection sensitivity for the virus. This new method's detection sensitivity of each virus was 10-100 times higher than that of ELISA using commercial kits and 1-10 times higher than that of one-step mRT-PCR with filter paper-based RNA extraction. The heat-treated homogenate with primers was also applicable for detecting eight other potato viruses by one-step conventional mRT-PCR and four potato viruses by real-time mRT-PCR which our research group previously developed. New direct RT-PCR method for potato viruses can be applied to seed certification programs, where a large number of samples need to be tested.
马铃薯易受病毒病影响，广泛的病毒感染会导致产量下降。马铃薯卷叶病毒、马铃薯S病毒、马铃薯X病毒和马铃薯Y病毒是马铃薯田中的重要病毒，在日本会造成重大经济损失。在本研究中，为了降低种子认证项目中病毒检测的 labor 和成本，开发了一种针对这些马铃薯病毒的简单分子诊断方法。该方法包括通过对叶片匀浆和新设计的引物组混合物进行热处理来制备模板，随后进行一步常规多重逆转录聚合酶链反应（mRT-PCR）。特别地，为了简化RNA提取，采用了一种程序，即将PBST中的叶片匀浆在无RNase水中稀释100倍，与引物一起在70°C下热处理5分钟，然后进行一步mRT-PCR。有趣的是，匀浆和引物混合物的热处理提高了对病毒的检测灵敏度。这种新方法对每种病毒的检测灵敏度比使用商业试剂盒的ELISA高10 - 100倍，比基于滤纸RNA提取的一步mRT-PCR高1 - 10倍。经引物热处理的匀浆也适用于通过一步常规mRT-PCR检测其他八种马铃薯病毒，以及通过我们研究小组先前开发的实时mRT-PCR检测四种马铃薯病毒。用于马铃薯病毒的新直接RT-PCR方法可应用于需要检测大量样品的种子认证项目。
