Recombinase Polymerase Amplification assays for detection of the major tropical root-knot nematodes

Detection of root-knot nematodes (RKN) in soil and plant samples is crucial to prevent its spread and select effective control measures. In this study, Recombinase Polymerase Amplification (RPA) assays using lateral flow dipsticks (LF-RPA) and real-time fluorescence detection (real-time RPA) were developed to detect the RKN species from tropical complex using a group-specific primer-probe set and Meloidogyne javanica using a species-specific primer-probe set. The results of the real time RPA assays in series of crude nematode extracts showed reliable detection within 16 min with a sensitivity of 1/100 of a second-stage juvenile in a reaction tube. The results of the LF-RPA assays showed reliable detection within 30 min with a sensitivity of 1/20 to 1/100 of a second-stage juvenile and 1/10 of a female in a reaction tube. Real-time RPA and LF-RPA assays are highly specific and can identify their target DNA in mixtures with other nematodes and plant tissues. LF-RPA assay has great potential for diagnosing RKN in the lab, field or in areas with a minimal laboratory infrastructure.