Rapid and sensitive detection of two fungal pathogens in soybeans using the recombinase polymerase amplification/ CRISPR‐Cas12a method for potential on‐site disease diagnosis

作者：Xiwen Sun, Rong Lei, Haipeng Zhang, W. Chen, Qianwen Jia, Xing Guo, Yongjiang Zhang, Pinshan Wu, Xinyi Wang · 发表于：Pest Management Science · 年份：2023 · DOI：10.1002/ps.7847 · 被引用次数：33 · 研究领域：Plant Pathogens and Fungal Diseases、Plant-Microbe Interactions and Immunity、Genomics and Phylogenetic Studies

BACKGROUND: Diaporthe aspalathi and Diaporthe caulivora are two of the fungal pathogens causing soybean stem canker (SSC) in soybean, which is one of the most widespread diseases in soybean growing regions and can cause 100% loss of yield. Current methods for the detection of fungal pathogens, including morphological identification and molecular detection, are mostly limited by the need for professional laboratories and staff. To develop a detection method for potential on-site diagnosis for two of the fungal pathogens causing SSC, we designed a rapid assay combining recombinase polymerase amplification (RPA) and CRISPR-Cas12a-based diagnostics to specifically detect D. aspalathi and D. caulivora. RESULTS: The translation elongation factor 1-alpha gene was employed as the target gene to evaluate the specificity and sensitivity of this assay. The RPA/CRISPR-Cas12a system has excellent specificity to distinguish D. aspalathi and D. caulivora from closely related species. The sensitivities of RPA/CRISPR-Cas12a-based fluorescence detection and lateral flow assay for D. aspalathi and D. caulivora are 14.5 copies and 24.6 copies, respectively. This assay can detect hyphae in inoculated soybean stems at 12 days after inoculation and has a recovery as high as 86% for hyphae-spiked soybean seed powder. The total time from DNA extraction to detection was not more than 60 min. CONCLUSION: The method developed for rapid detection of plant pathogens includes DNA extraction with magnetic b...