Detection of Sri Lankan cassava mosaic virus by loop-mediated isothermal amplification using dried reagents

Ayaka Uke; Sophary Khin; Kohei Kobayashi; Takuma Satou; Ok-Kyung Kim; Trinh Xuan Hoat; Keiko T Natsuaki; Masashi Ugaki

doi:10.1016/j.jviromet.2021.114336

Detection of Sri Lankan cassava mosaic virus by loop-mediated isothermal amplification using dried reagents

J Virol Methods. 2022 Jan:299:114336. doi: 10.1016/j.jviromet.2021.114336. Epub 2021 Oct 14.

Authors

Ayaka Uke¹, Sophary Khin², Kohei Kobayashi³, Takuma Satou³, Ok-Kyung Kim³, Trinh Xuan Hoat⁴, Keiko T Natsuaki⁵, Masashi Ugaki⁶

Affiliations

¹ Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa City, Chiba, 277-8562, Japan. Electronic address: uke@affrc.go.jp.
² National University of Battambang (UBB), National Road 5, Sangkat Preaek Preah Sdach, Battambang City, Battambang Province, 02352, Cambodia.
³ Department of Agricultural Science, Graduate School of Agriculture, Tokyo University of Agriculture, 1737 Funako, Atsugi, Kanagawa, 243-0034, Japan.
⁴ Plant Protection Research Institute (PPRI), Duc Thang Ward, Bac Tu Liem District, Ha Noi, 100000, Viet Nam.
⁵ Department of International Agricultural Development, Graduate School of Agriculture, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya, Tokyo 156-8502, Japan.
⁶ Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa City, Chiba, 277-8562, Japan.

PMID: 34656701
DOI: 10.1016/j.jviromet.2021.114336

Abstract

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.

Keywords: Dried reagent; LAMP; On-site detection; SLCMV.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Begomovirus* / genetics
Indicators and Reagents
Manihot*
Molecular Diagnostic Techniques
Nucleic Acid Amplification Techniques
Plant Diseases

Substances

Indicators and Reagents

Supplementary concepts

LAMP assay
Sri Lankan cassava mosaic virus