Rapid detection of the rice false smut fungus Ustilaginoidea virens by lateral flow strip‑based recombinase polymerase amplification assay

基于重组酶聚合酶扩增技术快速检测稻曲病菌

Jiacheng Xi^1*, Sanlian Wan^2*, Yue Li¹, Yuandi Xu¹, Jing Yang¹, Ting Zhang¹, Jiajia Chen³, Zhengguang Zhang¹, Danyu Shen^1#, Haifeng Zhang^1#

1 Sanya Institute of Nanjing Agricultural University/Key Laboratory of Integrated Management of Crop Diseases and Pests of Ministry of Education, Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China

2 Sanya Extension Service Center for Agricultural Technology, Sanya 572024, China

3 College of Landscape Architecture, Jiangsu Vocational College of Agriculture and Forestry, Zhenjiang 212400, China

摘要

稻曲病菌引起的稻曲病是水稻穗部的重要真菌病害之一。由于其侵染时期和部位的特殊性，导致稻曲病防治困难。因此，建立稻曲病的早期诊断与监测技术对阻断病害的发生与传播具有重要意义。重组酶聚合酶扩增技术（LF-RPA）具有省时、简便、假阳性率低、结果可视化等优点。本研究利用比较基因组学方法鉴定到稻曲病菌的特异基因Uv_3611，将其作为检测靶标，通过引物和探针的设计与筛选，建立了稻曲病菌的LF-RPA快速检测技术。该技术方法能特异性的检测出稻曲病菌，且在最佳反应条件下，检测的最低灵敏度为10 pg基因组DNA。进一步结合简易的PEG-NaOH核酸提取方法，无需特定的仪器设备，该技术在30分钟内即可快速检测出田间水稻各部位和商品化种子中的稻曲病菌。该检测方法可用于田间稻曲病的快速诊断与早期监测，同时可为及时、有效的稻曲病防控策略的制定提供技术支撑。

Abstract 

Rice false smut, caused by Ustilaginoidea virens, is a devastating disease that greatly reduces rice yield and quality.  However, controlling rice false smut disease is challenging due to the unique infection mode of U. virens.  Therefore, there is a need for early diagnosis and monitoring techniques to prevent the spread of this disease.  Lateral flow strip-based recombinase polymerase amplification (LF-RPA) overcomes the limitations of current U. virens detection technologies, which are time-consuming, require delicate equipment, and have a high false-positive rate.  In this study, we used a comparative genomics approach to identify Uv_3611, a specific gene of U. virens, as the target for the LF-RPA assay.  The designed primers and probe efffectively detected the genomic DNA (gDNA) of U. virens and demonstrated no cross-reactivity with related pathogens.  Under optimal conditions, the LF-RPA assay demonstrated a sensitivity of 10 pg of U. virens gDNA.  Additionally, by incorporating a simplified PEG-NaOH method for plant DNA extraction, the LF-RPA assay enabled the detection of U. virens in rice spikelets within 30 min, without the need for specialized equipment.  Furthermore, the LF-RPA assay successfully detected U. virens in naturally infected rice and seed samples in the field.  Therefore, the LF-RPA assay is sensitive, efficient, and convenient, and could be developed as a kit for monitoring rice false smut disease in the field.

全文链接：https://www.sciencedirect.com/science/article/pii/S2095311923003271

Journal of Integrative Agriculture (《农业科学学报》(英文), JIA) 由中华人民共和国农业农村部主管，中国农业科学院与中国农学会主办，中国农业科学院农业信息研究所承办。综合性英文学术期刊，月刊。创刊于2002年，现任主编为中国科学院院士陈化兰。JIA主要栏目有作物科学、园艺、植物保护、动物科学、动物医学、农业生态环境、食品科学、农业经济与管理等。刊稿类型有综述、研究论文、简报以及评述等。全部论文在Elsevier-ScienceDirect (SD) 平台OA出版。最新SCI影响因子4.6，位于SCI-JCR农业综合学科Q1区。中国科学院分区农林科学1区。2016年以来先后获得中国科协等部委 “提升计划”“登峰计划”“卓越计划”项目支持。