Detection of Sri Lankan cassava mosaic virus by loop-mediated isothermal amplification using dried reagents
2022
Uke, Ayaka | Khin, Sophary | Kobayashi, Kohei | Satou, Takuma | Kim, Ok-Kyung | Hoat, Trinh Xuan | Natsuaki, Keiko T. | Ugaki, Masashi
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.
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