苜蓿轮枝菌RPA/CRISPR-Cas12a快速检测方法的建立  

Establishment of RPA/CRISPR-Cas12a based rapid detection for Verticillium alfalfae

作　　者：李茗乐 雷荣[2] 杨藜 孙夕雯 杨益芬 王新一 范在丰 段维军 吴品珊[2] LI Mingyue;LEI Rong;YANG Li;SUN Xiwen;YANG Yifen;WANG Xinyi;FAN Zaifeng;DUAN Weijun;WU Pinshan(Key Laboratory of Surveillance and Management for Plant Quarantine Pests Ministry of Agriculture and Rural Affairs/College of Plant Protection,China Agricultural University,Beijing 100093,China;Chinese Academy of Inspection and Quarantine,Beijing 100176,China;Ningbo Institute of Inspection and Quarantine Science and Technology,Ningbo 315012,Zhejiang,China;Chengdu Customs Technology Centre,Chengdu 610041,Sichuan,China;Dalian University,Dalian 116622,Liaoning,China)

机构地区：[1]中国农业大学植物保护学院农业农村部植物检疫性有害生物监测防控重点实验室,北京100093 [2]中国检验检疫科学研究院,北京100176 [3]宁波检验检疫科学技术研究院,浙江宁波315012 [4]成都海关技术中心,四川成都610041 [5]大连大学,辽宁大连116622

出　　处：《菌物学报》2024年第9期80-95,共16页Mycosystema

基　　金：国家重点研发计划(2021YFC2600400,2021YFC2600402);中国检验检疫科学研究院基本科研业务费项目(2022JK20)。

摘　　要：苜蓿轮枝菌Verticillium alfalfae是我国重要的进境植物检疫对象,能够对十几种作物造成严重危害。为了快速检测苜蓿轮枝菌,本研究选用苜蓿轮枝菌的特征序列扩增区域[sequence characterized amplified region(SCAR)marker]作为目标基因,设计了特异性引物Va-RPA-F/Va-RPA-R和CRISPR引导RNA(Va-crRNA),建立了重组酶聚合酶等温扩增技术结合CRISPR-Cas12a的快速检测方法。对该检测体系的反应时间、组分浓度等进行优化,结果显示苜蓿轮枝菌基因组DNA在39℃恒温条件下扩增30 min后,其扩增产物用基于CRISPR-Cas12a的荧光法和侧向流层析试纸条两种方法检测,可检测到10.6pg基因组DNA,其灵敏度与实时荧光定量PCR结果相当,加标回收实验结果证明本研究建立的方法可成功用于植物组织中苜蓿轮枝菌的快速检测。Verticillium alfalfae is an important quarantine pathogen,which can cause serious damage to more than a dozen crops.The sequence characterized amplified region(SCAR)marker of V.alfalfae was chosen as the target gene,and a detection method combining RPA isothermal amplification technique and CRISPR-Cas12a system was established for specific detection of V.alfalfae.Under the optimized experimental conditions,10.6 pg genomic DNA can be detected by using CRISPR-Cas12a based fluorescence and lateral flow strip method after the genomic DNA was amplified at 39℃ for 30 minutes.The spiked recovery experiment indicated that the established RPA/CRISPR-Cas12a can be used for the rapid detection of V.alfalfae in plant tissues.

关 键 词：苜蓿轮枝菌 重组酶聚合酶等温扩增 CRISPR-Cas12a检测系统 快速检测 

分 类 号：S41[农业科学—植物保护]