假禾谷镰孢RPA-CRISPR/Cas12a快速检测体系的建立与应用  

Development and application of rapid detection system for Fusarium pseudograminearum based on RPA-CRISPR/Cas12a

作　　者：卫艳珂 王强 徐向明 胡小平[1] 商文静[1] WEI Yanke;WANG Qiang;XU Xiangming;HU Xiaoping;SHANG Wenjing(Key Laboratory of Plant Protection Resources and Pest Management of Ministry of Education,Key Laboratory of Integrated Pest Management on Crops in Northwestern Loess Plateau of Ministry of Agriculture and Rural Affairs,College of Plant Protection,Northwest A&F University,Yangling 712100,Shaanxi,China;Pest&Pathogen Ecology,NIAB East Malling,West Malling,Kent ME196BJ,UK)

机构地区：[1]西北农林科技大学植物保护学院,植保资源与病虫害治理教育部重点实验室,农业农村部西北黄土高原作物有害生物综合治理重点实验室,陕西杨凌712100 [2]英国国家农业植物研究院东茂林研究所病虫害生态学研究组,英国肯特郡ME196BJ

出　　处：《菌物学报》2024年第8期168-176,共9页Mycosystema

基　　金：国家重点研发计划项目(2022YFD1400101-1);科技部国际合作项目(G2023172013L);科技部对发展中国家科技援助项目(KY202002018);国家现代农业产业技术体系专项(CARS-03-37);农业农村部专项(152307101)。

摘　　要：假禾谷镰孢菌Fusarium pseudograminearum是引起我国黄淮麦区小麦茎基腐病的优势病原菌,对其精准、快速的检测有助于小麦茎基腐病的监测预警和精准防控。本研究基于F.pseudograminearum的NPS15基因,建立了重组酶聚合酶等温扩增技术(RPA)结合CRISPR/Cas12a的快速检测体系,该体系在37℃恒温条件下,40 min内可完成对目标病原菌的检测,检测灵敏度可达10^(–3)pg/μL,检测结果可通过核酸试纸条颜色反应直观读取,具有特异、灵敏、快速等优点。经验证,该体系可从带菌土壤、种子以及发病植株等复杂样本中检测到目标病原菌,为在田间实时快速检测F.pseudograminearum提供了一种高效方法。Fusarium pseudograminearum is the dominant pathogen causing wheat crown rot in the Huanghuai wheat-growing region in China.Accurate and rapid detection techniques are beneficial for monitoring and early warning of wheat crown rot.In this study,a rapid detection system combining RPA(recombinase polymerase amplification)with CRISPR/Cas12a was established based on the NPS15 gene of F.pseudograminearum,which can complete the detection of the target pathogen in 40 min at a constant temperature of 37℃,with a sensitivity of 10^(–3)pg/μL.The test results can be visually interpreted through the color reaction of nucleic acid test strips,with advantages of specificity,sensitivity,and rapidity.The system was validated to be able to detect the target pathogen from complex samples such as soil,seeds,and infected plants,providing an efficient method for the rapid detection of F.pseudograminearum in the field.

关 键 词：快速检测 重组酶聚合酶等温扩增 CRISPR/Cas12a检测体系 假禾谷镰孢菌 

分 类 号：S435.121.4[农业科学—农业昆虫与害虫防治]