Dual-target RPA-LFD/RF-RPA combined detection system: A rapid method for detecting the pathogens causing cowpea wilt disease.

Cowpea wilt disease, caused by the combined infection of Fusarium oxysporum and Phytophthora vignae, poses a persistent threat to crop yields and food security, driving the need for a dual-target detection method for early warning and informed crop management. This study developed a new method that can simultaneously detect two target pathogens, using multiplex recombinase polymerase amplification (RPA), followed by detection via lateral flow dipsticks (LFD) and real-time fluorescence (RF). The optimized system detects pathogens at 10 pg/mL within 20 minutes, with results visible via test strips or ultraviolet (UV) light. The system demonstrated a 100-fold increase in sensitivity over conventional PCR and reduced the detection time by 40 minutes. Field sample testing confirmed 100% specificity. This dual-target detection method presents a robust field-deployable solution, enabling rapid, sensitive, and equipment-free on-site diagnosis of cowpea wilt disease, which is critical for early prevention and boasts significant potential for practical application.