A Rapid, Equipment-Free Method for Detecting Avirulence Genes of Using a Lateral Flow Strip-Based RPA Assay

Rice blast, caused by , is one of the most destructive rice diseases worldwide. Using resistant rice varieties is the most cost-effective way to control rice blast. Consequently, it is critical to monitor the distribution frequency of avirulence () genes in rice planting fields to facilitate the breeding of resistant rice varieties. In this study, we established a rapid recombinase polymerase amplification-lateral flow dipstick (RPA-LFD) detection system for the identification of , , and . The optimized reaction temperature and duration were 37°C and 20 min, indicating that the reaction system could be initiated by body temperature without relying on any precision instruments. Specificity analysis showed that the primer and probe combinations targeting the three genes exhibited a remarkable specificity at genus-level detection. Under the optimized condition, the lower detected thresholds of , , and were 10 fg/μl, 100 fg/μl, and 10 pg/μl, respectively. Notably, the detection sensitivity of the three genes was much higher than that of PCR. In addition, we also successfully detected the presence of , , and in the leaf and panicle blast lesions with the RPA-LFD detection system. In particular, the genomic DNA was extracted using the simpler PEG-NaOH rapid extraction method. In summary, we developed an RPA detection system for , , and , combined with the PEG-NaOH rapid DNA extraction method. The innovative approach achieved rapid, real-time, and accurate detection of the three genes in the field, which is helpful to understand the distribution frequency of the three genes in the field and provide theoretical reference for the scientific layout of resistant rice varieties.