Recombinase polymerase amplification assay for sensitive and rapid detection of invasive fall armyworm, Spodoptera frugiperda

The fall armyworm, Spodoptera frugiperda, is an invasive, polyphagous pest that threatens approximately 353 plant species across 72 families worldwide. Due to morphological similarities with other noctuid pests during the early larval, pupal, and adult stages, traditional identification methods are labour-intensive and require specialist expertise. Rapid, reliable detection is essential given the pest's potential for widespread destruction. Through genome-wide in-silico analysis, this study identified a unique region within a signal peptide gene of S. frugiperda, which served as the basis for developing PCR, LAMP, and RPA-based assays for detection. The PCR assay produced a specific 550 bp amplicon for S. frugiperda, showing no cross-reactivity with negative controls. In the LAMP assay, positive samples exhibited a sky-blue colour, while negative samples turned violet when hydroxynaphthol blue dye was used. The RPA assay, with SYBR green dye, displayed bright green in positive samples and brick-red in negatives. Sensitivity tests demonstrated that PCR detected as low as 1 pg/µL, while LAMP and RPA achieved a higher sensitivity of 100 fg/µL. This study introduces the first RPA colorimetric assay for S. frugiperda, providing a time-efficient, cost-effective option that requires minimal equipment, ideal for field detection, thereby supporting timely pest monitoring and management.