Detection ofMesta yellow vein mosaic virus(MeYVMV) infield samples by aloop-mediated isothermal amplification reaction

Abstract

A loop-mediated isothermal amplification (LAMP) assay was optimized for the detection of Mesta yellow vein mosaic virus (MeYVMV) in diseased plants of mesta (Hibiscus sabdariffa L.& H. cannabinus L.). The LAMP assay was optimized using a set of six primers targeting the MeYVMV genome and could be completed in 30-60 min at 63 °C. The LAMP amplification results were visualized by adding 1 μl of hydroxy naphthol blue (HNB) dye in a 25 μl LAMP reaction mixture prior to amplification as well as by electrophoresis. The LAMP assay, which detected MeYVMV in a 10-5-fold diluted total DNA, was more sensitive than the PCR assay (10-4-fold dilution). The optimized LAMP assay was able to detect MeYVMV in different parts of the kenaf and roselle plants. Similarly, the optimized PCR assay was also capable of detecting MeYVMV in all the different parts of the kenaf plant but failed to detect the virus in the stem and flower buds of the roselle plant. Validation of the LAMP and LAMP with HNB dye assays revealed that the optimized reactions can be used successfully for the in-situ detection of MeYVMV in field samples and in virus quarantine programs. This is the first report of the detection of the begomovirus species, MeYVMV, in the mucilaginous plant species, kenaf and roselle, using a LAMP assay.