Rapid detection of fig mosaic virus using reverse transcription loop-mediated isothermal amplification
2015
Ishikawa, K. (The University of Tokyo, Bunkyo-ku, Tokyo (Japan). Graduate School of Agricultural and Life Sciences, Department of Agricultural and Environmental Biology, Laboratory of Plant Pathology) | Maejima, K. | Netsu, O. | Fukuoka, M. | Nijo, T. | Hashimoto, M. | Takata, D. | Yamaji, Y. | Namba, S.
Fig mosaic virus (FMV), a negative-strand RNA virus, is a causal agent of fig mosaic disease, which occurs in almost all countries where figs are grown and severely affects worldwide fig production. Reverse transcription-polymerase chain reaction (RT-PCR) using FMV-specific primers has typically been used to detect FMV, but faster and easier detection methods for FMV are required because RT-PCR requires multiple steps and special equipment. In this study, we established a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method to detect FMV. We designed LAMP primer sets based on sequence conservation among FMV isolates. The RT-LAMP reaction using a primer set targeting RNA3 in total RNA extracted from FMV-infected fig leaves resulted in rapid detection within 15 min. In addition, we established a fast and simple method of direct sampling from fig leaves using a wooden toothpick. The RT-LAMP reaction specificity and reactivity when using the direct sampling method were almost comparable to those obtained using isolated total RNA. Moreover, geographically and phylogenetically distinct FMV isolates were detectable using the FMV RT-LAMP assay. The assay presented here provides a practical method to detect FMV.
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