An advanced approach for rapid visual identification of <i>Spodoptera frugiperda</i> (Lepidoptera: Noctuidae) based on multienzyme isothermal rapid amplification with lateral flow dipstick assay in China

Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) is highly invasive lepidopteran pest of global significance. Differentiating S. frugiperda from similar species, especially in non-adult stages, remains challenging. Rapid molecular identification techniques, such as multienzyme isothermal rapid amplification (MIRA) combined with lateral flow dipstick (MIRA-LFD), play a crucial role in early monitoring and safeguarding agricultural production. Our study introduces this platform for the rapid visual identification of S. frugiperda. The specific MIRA primers and the LFD probe were designed based on the COX1 genes. The MIRA reaction conditions were optimized (at 37 °C for 15 min) for effective template DNA amplification. The MIRA-LFD system achieved precise identification of the target species within 12 min at 37 °C. This method exhibited high specificity and sensitivity, allowing for detection from 1.0 ng·μl-1 of DNA. Combined with rapid DNA extraction, expedited identification of individual S. frugiperda at different developmental stages was achieved, enhancing the practicality and convenience of the established methods. In summary, our research demonstrates that the MIRA-LFD method for S. frugiperda detection achieved high accuracy with rapid results (entire workflow completion within 22 min at 37 °C, including crude DNA extraction, MIRA amplification, and LFD dipstick interpretation). This approach does not require expensive equipment, making it highly practical for field applications. It offers an improved solution for early detection of invasive arthropod pests and enhances field surveillance capabilities, thereby advancing precision pest management strategies.