Rapid detection of Meloidogyne spp. by LAMP assay in soil and roots

Loop-mediated isothermal amplification (LAMP), a novel DNA amplification technique, has been used to detect a variety of pathogens including viruses, fungi, bacteria and parasites. However, diagnosis of sedentary plant-parasitic nematode (PPN) species has not yet been attempted. In this study, we developed a universal LAMP set (RKN-LAMP) for the diagnosis of four common Meloidogyne species (Meloidogyne incognita, M. arenaria, M. javanica and M. hapla), and M. incognita-specific LAMP set (Mi-LAMP). In both assays, a typical ladder-like pattern on gel electrophoresis was observed in all positive samples but not in the negative controls. Amplification products were further confirmed using restriction analysis of the Hpa II enzyme, detection by visual inspection using SYBR Green I and the lateral flow dipstick (LFD) assay. The two LAMP sets were specifically able to detect four common Meloidogyne species and M. incognita populations having several different geographical origins and pathotypes. No cross reaction with DNA of other PPNs was observed. Sensitivity of the RKN- and Mi-LAMP was 10 and 100 fg of pure genome DNA respectively. Both LAMP sets could also amplified crude DNA isolated from the galled root tissue and from soil containing juveniles of M. incognita. The RKN- and Mi-LAMP sets offer the advantages of simplicity, rapidity and cost effectiveness. Both LAMP sets will be instrumental for the diagnosis of Meloidogyne spp. by local extension and regulatory personnel.

The Lateral flow dipstick tests shown that the RKN- and Mi-LAMP detection were specific for common Meloidogyne species and M. incognita respectively.Figure optionsDownload as PowerPoint slideHighlights
► For the first time, we developed LAMP sets for the diagnosis of Meloidogyne species.
► The LAMP amplification results could visually inspected by naked eye.
► The developed LAMP sets could amplify crude DNA isolated from the root and the soil samples.