Lateral Flow Devices (LFDs) provide a rapid and practical method for detecting foot-and-mouth disease (FMD), which is particularly valuable in endemic regions with limited laboratory infrastructure. Previous prototypes allowed simple FMD confirmation, but their capacity to identify specific serotypes—critical for understanding disease spread and guiding vaccine strategies—was limited. This work reports on the validation of two multiplex LFDs constructed using well-characterized monoclonal antibodies: LFD1, designed to detect serotypes O, A, and Asia1, and LFD2, which identifies SAT1 and SAT2. To assess their analytical breadth, 83 cultured FMDV strains were tested with LFD1 and 15 strains with LFD2, demonstrating that both devices could detect and distinguish multiple viral lineages across all seven endemic pools. The diagnostic sensitivity of viral detection was evaluated using 224 real-time RT-PCR–positive field samples, showing 87.5% detection (140/160) for LFD1 and 70% (45/64) for LFD2. For serotyping accuracy, the devices were compared with an antigen-ELISA. LFD1 correctly typed 96% of O-positive samples (n = 95), 94% of A-positive samples (n = 54), and 83% of Asia1-positive samples (n = 29) from 178 field samples. LFD2 achieved 77% accuracy for SAT1 (n = 52) and 82% for SAT2 (n = 40) among 92 positive samples. Testing 60 epithelium homogenates from FMD-free animals confirmed 100% specificity. These results suggest that the multiplex LFDs could serve as reliable field-deployable tools, supporting rapid FMD surveillance and aiding in outbreak management.
