Construction of a Field-Oriented Point-of-Care Testing System Based on MIRA-LFIA for Detection of Puccinia polysora DNA in Asymptomatic Maize Leaves

Southern corn rust, caused by Puccinia polysora Underw, is a major foliar disease that threatens corn yield in China. Detection of P. polysora during the corn growing period is critical for timely intervention and disease management. In this study, specific primers and probes were designed based on the variable region of the rDNA-ITS sequence. After conventional PCR validation, specificity screening, and sensitivity evaluation, an optimal primer–probe combination was successfully established. The developed assay achieved a detection limit of 1 pg·μL−1 genomic DNA and a DNA equivalent of 57.6 urediniospores of P. polysora. The reaction conditions were further optimized, and stable amplification could be completed under a constant temperature of 38 °C for 15 min. The developed MIRA-LFIA was evaluated using 70 field maize leaf samples, with the reported OTNPCR (One-Tube-Nested PCR) method used as the gold-standard reference. MIRA-LFIA produced 55 positive and 15 negative results, whereas OTNPCR yielded 57 positive and 13 negative samples. A diagnostic evaluation revealed that the MIRA-LFIA assay achieved a sensitivity of 96.49%, specificity of 100%, positive predictive value of 100%, negative predictive value of 86.67%, and Youden’s index of 0.965., with a Kappa value of 0.911. This MIRA-LFIA system enables rapid detection and visual interpretation of southern corn rust. It features simple operation and low technical requirements, making it suitable for rapid on-site monitoring at grassroots institutions and in fields.