植物保护学报2025，Vol.52Issue(4)：812-821,10.DOI:10.13802/j.cnki.zwbhxb.2025.2025042
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
Rapid detection method for rice brown leaf spot pathogen Bipolaris oryzae based on RPA/CRISPR-Cas12b technology
摘要
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 detec-tion 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/RPA-BoAra43A-R).Subsequently,the pathogen could be specifically detected using both CRISPR-Cas12b-based 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,on-site detection of field-collected rice leaf samples and could be completed within one hour,demonstrat-ing its simplicity and practicality for field applications.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Key words
Bipolaris oryzae/CRISPR-Cas12b/recombinase polymerase amplification/fluorometric assay/lateral flow strip assay引用本文复制引用
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[J].植物保护学报,2025,52(4):812-821,10.基金项目
内蒙古自治区科技"突围"工程项目(2025KJTW0029) （2025KJTW0029）
