Development of a dual-mode loop-mediated isothermal amplification genotyping system for rapidly detecting EPSPS mutations in glyphosate-resistant goosegrass ( Eleusine indica )

Abstract Goosegrass [ Eleusine indica (L.) Gaertn.], one of the most troublesome weeds threatening global crop production, has developed resistance to glyphosate, a widely used herbicide that targets 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; HRAC Group 9). The most commonly reported EPSPS mutations in glyphosate-resistant E. indica include Pro-106-Leu (P106L), Pro-106-Ser (P106S), and a double mutation of Thr-102-Ile with Pro-106-Ser (T102I + P106S). Although conventional methods such as polymerase chain reaction (PCR) and (derived) cleaved amplified polymorphic sequence [(d)CAPS] are widely used for mutation detection, their applicability remains limited because they are expensive, time-consuming, and technically complex. We developed a dual-mode loop-mediated isothermal amplification (LAMP) genotyping system for the rapid detection of three key glyphosate-resistance mutations (P106L, P106S, and T102I) in E. indica . The system integrated a closed-tube colorimetric assay for on-site screening and a real-time fluorescence ΔCt threshold analysis for laboratory-based quantification. Dual primer sets were designed to distinguish homozygous, heterozygous, and susceptible genotypes while preventing aerosol contamination. Validation using 150 samples demonstrated accuracies of 90% (colorimetric) and 92% (fluorescent). Sensitivity analysis revealed a 1,000-fold improvement over conventional PCR (detection limit: 5 × 10 −4 vs. 5 × 10 −1 ng μl −1 ). The closed-tube design eliminated contamination risks, and the ΔCt threshold enabled precise heterozygote identification. This cost-effective, time-saving, and high-precision system provides a robust tool for the early monitoring of glyphosate resistance in E. indica , guiding efficient control and mitigation of resistance spread in crop fields.