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

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.