丁香疫霉菌RPA/CRISPR-Cas12a快速检测方法的建立  

Development of rapid detection for Phytophthora syringae based on RPA/CRISPR-Cas12a

作　　者：雷荣[1] 孙夕雯 江丽[1] 王振华 李国庆[4] 李远[5] 廖晓玲 吴品珊[1] Lei Rong;Sun Xiwen;Jiang Li;Wang Zhenhua;Li Guoqing;Li Yuan;Liao Xiaoling;Wu Pinshan(Chinese Academy of Inspection and Quarantine,Beijing 100176,China;College of Land and Environment of Shenyang Agricultural University;Wuhan Customs District;Huazhong Agriculture University;Central Laboratory of Yongchuan Hospital,Chongqing Medical University;Chongqing University of Science and Technology)

机构地区：[1]中国检验检疫科学研究院,北京100176 [2]沈阳农业大学土地与环境学院 [3]武汉海关 [4]华中农业大学 [5]重庆医科大学永川附属医院 [6]重庆理工大学

出　　处：《植物检疫》2022年第3期31-38,共8页Plant Quarantine

基　　金：国家重点研发计划项目(2021YFC2600402);中国检验检疫科学研究院基本科研业务费项目(2020JK048)。

摘　　要：丁香疫霉病菌(Phytophthora syringae,PS)造成数十种蔷薇科植物严重病害,是我国的植物检疫性有害生物。本研究根据GenBank中PS的Ras-like protein(Ypt1)基因,建立重组酶聚合酶等温扩增结合CRISPR-Cas12a系统的荧光法和侧向流层析试纸条快速检测方法,以实现在田间或口岸快速、准确、灵敏检测丁香疫霉病菌的目的。本研究优化了RPA/CRISPR-Cas12a的反应条件,考察了特异性、灵敏度以及实际样品检测能力。结果表明,该方法37℃扩增40 min,能特异性地检测丁香疫霉菌,灵敏度为133 fg,与荧光定量PCR相当。本研究建立的快速检测方法可用于丁香疫霉菌的快速诊断。Phytophthora syringae(PS)causes serious diseases of dozens of Rosaceae plants and is a plant quarantine pest in China.In this study,based on the Ras-like protein(Ypt1)gene of PS in NCBI database,we developed a combined method of recombinase polymerase amplification(RPA)and CRISPR-Cas12a system(RPA/CRISPR-Cas12a)with fluorescent reporter and lateral flow reporter,in order to realize the rapid,specific and sensitive detection of PS in field or at port.The reaction conditions of RPA/CRISPR-Cas12a were optimized,and the specificity,sensitivity and applicability of real samples were investigated.The results showed that this RPA/CRISPR-Cas12a can specifically detect PS with a sensitivity of 133 fg PS genomic DNA at 37℃within 40 min.The rapid detection method established in this study can be used for rapid field diagnosis of Phytophthora syringae.

关 键 词：丁香疫霉病菌 重组酶聚合酶扩增 CRISPR-Cas12a 快速检测 

分 类 号：S432.44[农业科学—植物病理学]