BACKGROUNDPantoea stewartii subsp. stewartii and Maize dwarf mosaic virus (MDMV) infections severely affect corn productivity worldwide. Rapid point‐of‐need diagnoses of quarantine pathogens P. stewartii subsp. stewartii and MDMV are required for early detection, timely disease management and ensuring phytosanitary regulations. Recombinase polymerase amplification (RPA) is an isothermal technique suitable for rapid diagnostics using minimally processed samples. Integrating CRISPR/Cas collateral activities with the RPA assays further enhances the specificity and sensitivity of molecular toolkits for diagnostic assays.RESULTSRPA‐CRISPR/Cas12a assay targeting the intergenic spacer region between capsular polysaccharide genes cpsA and cpsB of P. stewartii subsp. stewartii detected 1 × 10−6 ng DNA μL−1 using real‐time fluorescence and blue light observation methods, and 1 × 10−4 ng DNA μL−1 using the lateral flow dipstick (LFD). Likewise, RPA‐CRISPR/Cas13a assay detected MDMV coat protein (CP) gene with an ultrasensitive detection limit of 3.69 × 10−7 ng μL−1 using the real‐time fluorescence and blue light observation methods, and 3.69 × 10−5 ng μL−1 using the LFD. The dual RPA‐CRISPR/Cas assays detected both pathogens without compromising the speed and/or detection sensitivity of the single assays.CONCLUSIONThe validated assays provide a useful and sensitive molecular tool for detecting two quarantine pathogens of maize within a minimal resource framework suitable for fast‐tracking the containment strategies. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

中文翻译：

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单和双 RPA-CRISPR/Cas 检测，用于玉米和玉米矮化花叶病毒的斯图尔特枯萎病原菌（Pantoea stewartii subsp. stewartii）的按需检测


背景 Pantoea stewartii subsp. stewartii 和玉米矮花叶病毒 （MDMV） 感染严重影响全球玉米生产力。需要对检疫病原体 P. stewartii subsp. stewartii 和 MDMV 进行快速需求诊断，以便早期发现、及时进行疾病管理和确保植物检疫法规。重组酶聚合酶扩增 （RPA） 是一种等温技术，适用于使用最少处理的样品进行快速诊断。将 CRISPR/Cas 侧支活性与 RPA 检测相结合进一步提高了分子工具包用于诊断检测的特异性和灵敏度。结果针对斯图尔蒂亚种荚膜多糖基因 cpsA 和 cpsB 之间的基因间隔区的 rpa-CRISPR/Cas12a 测定使用实时荧光和蓝光观察方法检测到 1 × 10-6 ng DNA μL-1，以及 1 × 10-4 ng DNA μL-1使用侧向层析试纸 （LFD）。同样，RPA-CRISPR/Cas13a 检测试剂盒使用实时荧光和蓝光观察方法检测 MDMV 外壳蛋白 （CP） 基因，超灵敏检测限为 3.69 × 10-7 ng μL-1，使用 LFD 检测 3.69 × 10-5 ng μL-1。双重 RPA-CRISPR/Cas 检测可检测两种病原体，而不会影响单次检测的速度和/或检测灵敏度。结论经过验证的检测提供了一种有用且灵敏的分子工具，用于在适合快速跟踪遏制策略的最小资源框架内检测玉米的两种检疫病原体。© 2024 作者。由John Wiley & Sons Ltd代表化学工业协会出版的《害虫管理科学》。