Uke Ayaka, Khin Sophary, Kobayashi Kohei, Satou Takuma, Kim Ok-Kyung, Hoat Trinh Xuan, Natsuaki Keiko T, Ugaki Masashi
Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa City, Chiba, 277-8562, Japan.
National University of Battambang (UBB), National Road 5, Sangkat Preaek Preah Sdach, Battambang City, Battambang Province, 02352, Cambodia.
J Virol Methods. 2022 Jan;299:114336. doi: 10.1016/j.jviromet.2021.114336. Epub 2021 Oct 14.
Recently, the widespread occurrence of Sri Lankan cassava mosaic virus (SLCMV), genus Begomovirus, family Geminiviridae, which causes a mosaic disease in cassava (Manihot esculenta Crantz) in South-East Asia have, become a serious economic issue. Since cassava is propagated through vegetative cuttings, a rapid virus diagnostic method is crucial for generating virus-free planting materials. In this study, a loop-mediated isothermal amplification (LAMP) assay using six primers was developed and validated for the rapid detection of SLCMV in cassava leaves. This SLCMV assay had a detection sensitivity that was up to 10,000 times higher than that of the conventional polymerase chain reaction assay and can detect the virus from symptomless stem cutting, which is a potential long-distance spreader of the virus. Furthermore, a practical LAMP protocol using stable dried reagents from a commercial kit was established so that the assay could be performed in the field by incubating the reactions in water at 60-65 °C instead of using a thermal cycler. The primer sequences and the LAMP protocol described here should be useful for the rapid and sensitive on-site detection of SLCMV.
最近，双生病毒科菜豆金色花叶病毒属的斯里兰卡木薯花叶病毒（SLCMV）在东南亚木薯（Manihot esculenta Crantz）上广泛发生，已成为一个严重的经济问题。由于木薯通过营养扦插繁殖，快速的病毒诊断方法对于生产无病毒种植材料至关重要。在本研究中，开发了一种使用六条引物的环介导等温扩增（LAMP）检测方法，并验证了其用于快速检测木薯叶片中SLCMV的有效性。这种SLCMV检测方法的检测灵敏度比传统聚合酶链反应检测方法高多达10000倍，并且能够从无症状的茎段插条中检测到病毒，而无症状茎段插条是该病毒潜在的远距离传播载体。此外，建立了一种使用来自商业试剂盒的稳定干燥试剂的实用LAMP方案，这样通过在60 - 65°C的水中孵育反应而不是使用热循环仪，该检测方法就可以在现场进行。这里描述的引物序列和LAMP方案对于快速、灵敏地现场检测SLCMV应该是有用的。
