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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。
Abstract：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.
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基本信息:
DOI：10.19662/j.cnki.issn1005-2755.2022.03.006
中图分类号:S41-30
引用信息:
[1]雷荣,孙夕雯,江丽,等.丁香疫霉菌RPA/CRISPR-Cas12a快速检测方法的建立[J].植物检疫,2022,36(03):31-38.DOI:10.19662/j.cnki.issn1005-2755.2022.03.006.
基金信息:
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）