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基于重组酶聚合酶扩增/CRISPR-Cas12b技术的水稻胡麻叶斑病病原菌快速检测方法

引用本文：郭凤,刘华容,吕世民,李艺霖,孙文献,汪激扬.基于重组酶聚合酶扩增/CRISPR-Cas12b技术的水稻胡麻叶斑病病原菌快速检测方法.植物保护学报,2025,52(4):812-821

DOI：10.13802/j.cnki.zwbhxb.2025.2025042

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作者	单位	E-mail
郭凤	中国农业大学三亚研究院, 海南三亚 572025 中国农业大学植物保护学院, 北京 100193
刘华容	中国农业大学三亚研究院, 海南三亚 572025 中国农业大学植物保护学院, 北京 100193
吕世民	中国农业大学三亚研究院, 海南三亚 572025 中国农业大学植物保护学院, 北京 100193
李艺霖	中国农业大学三亚研究院, 海南三亚 572025 中国农业大学植物保护学院, 北京 100193
孙文献	中国农业大学三亚研究院, 海南三亚 572025 中国农业大学植物保护学院, 北京 100193 吉林农业大学植物保护学院, 长春 130118	wxs@cau.edu.cn
汪激扬	中国农业大学三亚研究院, 海南三亚 572025 中国农业大学植物保护学院, 北京 100193	aqwjy@cau.edu.cn

中文摘要:为实现水稻胡麻叶斑病病原菌稻平脐蠕孢菌Bipolaris oryzae的快速检测，以稻平脐蠕孢菌基因BoAra43A的特征序列为靶标，利用重组酶聚合酶扩增（recombinase polymerase amplification，RPA）试剂盒筛选特异性引物，并根据靶基因序列设计CRISPR单链向导RNA（single guide RNA，sgRNA），构建结合RPA技术与CRISPR-Cas12b系统的稻平脐蠕孢菌快速检测方法。结果显示：在40℃恒温条件下，利用筛选到的特异性引物RPA-BoAra43A-F/RPA-BoAra43A-R对稻平脐蠕孢菌基因组DNA扩增30 min后，使用基于CRISPR-Cas12b的荧光法和侧流层析试纸条法均可特异性检测到稻平脐蠕孢菌，且对其同属病原菌及水稻常见病原菌无假阳性。此外，荧光法和侧流层析试纸条法的检测极限为0.43 ng/μL基因组DNA，与传统PCR处于同一数量级。利用所建试纸条法可在1 h内完成田间水稻叶片样品的检测，具有操作简便和现场快速检测的优势。

中文关键词:稻平脐蠕孢菌 CRISPR-Cas12b 重组酶聚合酶扩增荧光法侧流层析试纸条法

Rapid detection method for rice brown leaf spot pathogen Bipolaris oryzae based on RPA/CRISPR-Cas12b technology

Author Name	Affiliation	E-mail
Guo Feng	Sanya Institute of China Agricultural University, Sanya 572025, Hainan Province, China College of Plant Protection, China Agricultural University, Beijing 100193, China
Liu Huarong	Sanya Institute of China Agricultural University, Sanya 572025, Hainan Province, China College of Plant Protection, China Agricultural University, Beijing 100193, China
Lü Shimin	Sanya Institute of China Agricultural University, Sanya 572025, Hainan Province, China College of Plant Protection, China Agricultural University, Beijing 100193, China
Li Yilin	Sanya Institute of China Agricultural University, Sanya 572025, Hainan Province, China College of Plant Protection, China Agricultural University, Beijing 100193, China
Sun Wenxian	Sanya Institute of China Agricultural University, Sanya 572025, Hainan Province, China College of Plant Protection, China Agricultural University, Beijing 100193, China College of Plant Protection, Jilin Agricultural University, Changchun 130118, Jilin Province, China	wxs@cau.edu.cn
Wang Jiyang	Sanya Institute of China Agricultural University, Sanya 572025, Hainan Province, China College of Plant Protection, China Agricultural University, Beijing 100193, China	aqwjy@cau.edu.cn

Abstract:To achieve rapid detection of Bipolaris oryzae, the causal agent of rice brown leaf spot, a target region was selected based on a characteristic sequence of BoAra43A gene. Specific primers were screened for a recombinase polymerase amplification (RPA) assay, and a CRISPR single guide RNA (sgRNA) was designed accordingly. By integrating RPA with the CRISPR-Cas12b system, a rapid detection method for B. oryzae was established. Under isothermal conditions at 40 ℃, genomic DNA of B. oryzae could be amplified within 30 min using the selected specific primers (RPA-BoAra43A-F/RPABoAra43A-R). Subsequently, the pathogen could be specifically detected using both CRISPR-Cas12bbased fluorescence assay and lateral flow strip assay, with no false positives observed for closely related fungal species or other common rice pathogens. Moreover, the detection limit of both methods was 0.43 ng/μL genomic DNA, comparable to conventional PCR. The lateral flow assay enabled rapid, onsite detection of field-collected rice leaf samples and could be completed within one hour, demonstrating its simplicity and practicality for field applications.

keywords:Bipolaris oryzae CRISPR-Cas12b recombinase polymerase amplification fluorometric assay lateral flow strip assay

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