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

Goosegrass [ Eleusine indica (L.) Gaertn.], one of the most troublesome weed threats to 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 sequences (dCAPS) are widely used for mutation detection, their applicability remains limited because of their high cost, time consumption, and technical complexity. 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). 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.