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

Xiaobo Sun,Rong Lei,Haipeng Zhang, W. Chen, Qianwen Jia, Xinfeng Guo,Yongjiang Zhang,Pinshan Wu,Xinyi Wang

Pest Management Science(2023)

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摘要
Abstract BACKGROUD 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, 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 (tef1) 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 the closely related species. The sensitivity 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 beads or rapid DNA extraction, isothermal nucleic acid amplification at 39°C, CRISPR‐Cas12a cleavage reaction at 37°C, and lateral flow assay or end point fluorescence visualization at room temperature. The RPA reagents and CRISPR‐Cas12a reagents can be pre‐loaded in the microcentrifuge tube to simplify the procedures in the field. Both RPA and CRISPR‐Cas12a reaction can be realized on a portable incubator, and the results are visualized using lateral flow strips or portable flashlight. This method requires minimal equipment and operator training, has promising applications for rapid on‐site disease screening, port inspection, or controlling fungal pathogen transmission in crop. This article is protected by copyright. All rights reserved.
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关键词
fungal pathogens,<scp>rpa</scp>/<scp>crispr‐cas12a</scp>,soybeans,sensitive detection
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