Rapid and Visual Detection of <i>Pyricularia oryzae</i> Using Coupled Recombinase Polymerase Amplification–Lateral Flow Dipstick Assay

Rice blast, caused by <i>Pyricularia oryzae</i>, is one of the most destructive fungal diseases in rice, severely impacting rice production worldwide every year. Rapid, accurate, and visual detection of <i>P. oryzae</i> is essential for more effective prevention and control. In this study, we developed a recombinase polymerase amplification-lateral flow dipstick (RPA-LFD) assay to detect <i>P. oryzae</i>. Species-specific RPA primer pairs and probe were designed based on the target gene MGG_15975. The optimized reaction temperature and time were set at 37°C and 25 min, respectively. Specificity analysis showed that the assay could specifically detect <i>P. oryzae</i> isolates from rice, whereas other fungal species or <i>Pyricularia</i> species from grasses were not detected. Additionally, this assay demonstrated high sensitivity, capable of detecting as low as 10^-2 ng/μl of <i>P. oryzae</i> genomic DNA, which was found to be 100 times more sensitive than conventional PCR. Furthermore, using this assay, <i>P. oryzae</i> was effectively detected in diseased leaves in rice fields and could also be identified at an early stage of infection before obvious lesions appeared in artificially inoculated rice seedlings. Therefore, the RPA-LFD assay developed in our study for the detection of <i>P. oryzae</i> is rapid, highly sensitive, and efficient, which has the potential application for early diagnosis of <i>P. oryzae</i> infection in rice fields.