Rapid and sensitive detection of the rice root-knot nematode Meloidogyne graminicola and screening of M. oryzae using recombinase polymerase amplification

Abstract

Meloidogyne graminicola (Mg), also known as the rice root-knot nematode (RRKN), is a devastating pest in rice production, leading to significant yield losses in affected regions. Therefore, early and accurate detection of Mg is critical for timely intervention and effective management. Although molecular DNA-based methods for Mg detection and identification are accessible and reliable, they require skilled technicians, specialized equipment, and expensive laboratory facilities, and are not suitable for on-site detection. In this study, a Recombinase Polymerase Amplification (RPA) assay was developed for the rapid and accurate detection of Mg targeting a Sequence Characterised Amplified Region (SCAR). Our optimized RPA assay -TwistAmp Basic and TwistAmp exo (TwistDx, Cambridge, UK) - successfully screened M. oryzae and identified Mg directly from crude extracts of second-stage juveniles (J2), eliminating the need for complex DNA extraction steps. Using a dilution series of crude nematode extracts combined with real-time fluorescent detection, our RPA assay achieved an accurate result within 8 min, successfully identifying a single second-stage juvenile (J2) of Mg in mixed populations containing non-target species at ratios as low as 1:9. Additionally, M. oryzae amplified with a significant delay (14 min) and remained below the baseline (50 dRn), allowing a clear distinction from Mg and serving as initial screening of this species. This ultra-fast, sensitive, and user-friendly method presents a game-changing tool for early and on-site detection of Mg, paving the way for more effective nematode management strategies in rice cultivation.