Detection of Mesta yellow vein mosaic virus (MeYVMV) in field samples by a loop-mediated isothermal amplification reaction

• Published in: Journal of virological methods 2019-01, Vol.263, p.81-87
• Main Authors: Meena, Prabhu Narayan, Kharbikar, Lalit Laxman, Rana, Rajeev Singh, Satpathy, Subrata, Shanware, Arti, Sivalingam, Palaiyur Nanjappan, Nandanwar, Shweta
• Format: Article
• Language: English
• Subjects: Begomovirus - genetics; Begomovirus - isolation & purification; Hibiscus - virology; Kenaf; LAMP; Mesta yellow vein mosaic virus; Naphthalenesulfonates; Nucleic Acid Amplification Techniques - standards; PCR; Plant Diseases - virology; Polymerase Chain Reaction; Reproducibility of Results; Roselle; Sensitivity and Specificity; Virus detection
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2018.10.016

•Diagnostics for MeYVMV in mesta (Hibiscus sabdariffa L. and H. cannabinus L.) by loop-mediated isothermal amplification assay was developed.•LAMP assay developed in this study for the detection of MeYVMV in mesta is more sensitive than PCR technique.•The LAMP assay has been optimized using Hydroxy Naphthol Blue (HNB) dye is more suitable for field sample diagnosis and in quarantine.•This is the first report for detection of begomovirus species, MeYVMV in the mucilaginous plant species, kenaf and roselle using LAMP. 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.