Detection of Kyuri Green Mottle Mosaic Virus from Soil by the Immunocapture Reverse Transcription Loop-mediated Isothermal Amplification Reaction

摘要

Kyuri green mottle mosaic virus (KGMMV) which is a major soil-borne virus, causes severe yield reduction in cucumber in Japan. We developed immunocapture reverse transcription loop-mediated isothermal amplification (Ic/RT-LAMP) method to detect KGMMV in soil. Degenerate primer set from KGMMV C strain (KGMMV-C) and Yodo strain (KGMMV-Yodo) was designed for the detection of both strains. To optimize the reaction condition, the RT-LAMP reaction using the degenerate primer set was performed at different temperature (60-65°C). The reaction at 60°C gave the best results. The result on specificity test using 7 cucumber pathogenic viral isolates indicated that the RT-LAMP assay established in this study had no cross reactions. The detection limit of the RT-LAMP assay was 10 pg of purified KGMMV. The sensitivity of RT-LAMP was 10 times higher than that of RT-PCR. Detection limit of DAS-ELISA and Ic/RT-LAMP was compared by using of the dilution of purified KGMMV-C in soil. Ic/RT-LAMP could detect 0.5 ng of KGMMV from 100 mg of soil sample and the sensitivity of Ic/RT-LAMP was 200 times higher than that of DAS-ELISA. The Ic/RT-LAMP assay could successfully detect KGMMV in soil samples from 10 fields where KGMMV had been detected in a previous cultivation. The present Ic/RT-LAMP method showed to be a rapid, simple and available detection method of KGMMV in soil.