RT-RAA-CRISPR/Cas12a可视化检测番茄褐色皱纹果病毒  

Visual detection of tomato brown rugose fruit virus by RT-RAA-CRISPR/Cas12a method

作　　者：赵振兴 范奇璇 王思元 董铮 胡中泽 周涛[2] 张永江[1] Zhao Zhenxing;Fan Qixuan;Wang Siyuan;Dong Zheng;Hu Zhongze;Zhou Tao;Zhang Yongjiang(Chinese A-cademy of Inspection and Quarantine,Beijing 100176,China;College of Plant Protection,China Agricultur-al University;Institute of Taizhou Agricultural Science,Jiangsu Academy of Agricultural Sciences)

机构地区：[1]中国检验检疫科学研究院,北京100176 [2]中国农业大学植物保护学院 [3]江苏省农业科学院泰州农科所

出　　处：《植物检疫》2024年第3期14-20,共7页Plant Quarantine

基　　金：国家重点研发计划(2021YFD1400100,2021YFD1400103)。

摘　　要：番茄褐色皱纹果病毒(tomato brown rugose fruit virus,ToBRFV)严重威胁番茄等茄科园艺作物的生产安全。本研究根据其外壳蛋白(coat protein,CP)基因及其同属病毒的差异序列,设计了特异性重组酶介导等温核酸扩增技术(recombinase-aided amplification,RAA)引物,并基于CRISPR/Cas12a的设计原则,设计了靶向RT-RAA扩增产物的CRISPR RNA(crRNA)。通过优化获得了检测信号最强的反应体系,其中报告基因FQ终浓度为600nmol/L、Cas12a和crRNA终浓度分别为200nmol/L和1000nmol/L,最终总反应时间仅为30 min。该方法可特异性检测ToBRFV,对携带ToBRFV的番茄样品RNA检测灵敏度为RT-PCR和RT-qPCR的10000和100倍,检测限为3.46fg/μL。阳性样品验证结果显示,本研究建立的RT-RAA-CRISPR/Cas12a检测技术可以在不同来源的辣椒、番茄侵染的植物叶片、果实及种子中检测到ToBRFV,表明该技术可用于番茄褐色皱纹果病毒的快速、灵敏的可视化检测。Tomato brown rugose fruit virus(ToBRFV)threatens production of Solanaceae crops like tomato.In this study we designed specific recombinase-aided amplification(RAA)primers for rapid isothermal amplification of ToBRFV based on its coat protein(CP)gene and the differential sequences of related viruses,and designed targeted CRISPR RNA(crRNA)targeting RT-RAA amplification products based on the design principle of CRISPR/Cas12a principles.The reaction took only 30 minutes to com-plete and resulting in the strongest visual detection signal,with a final concentration of reporter gene FQ,Cas12a,and crRNA of 600 nmol/L,200 nmol/L,and 1000 nmol/L in the reaction system,respectively.This established RT-RAA-CRISPR/Cas12a assay was able to specific detect ToBRFV with a detection limit of 3.46 fg/μL,which was 10000 and 100-fold higher than conventional RT-PCR and RT-qPCR methods.Val-idation test results for leaf,fruit,and seed samples from chili pepper and tomato plants of different ori-gins suggest that the RT-RAA-CRISPR/Cas12a detection method can be used for rapid and sensitive visu-al detection of ToBRFV.

关 键 词：番茄褐色皱纹果病毒 RT-RAA CRISPR/Cas12a 可视化检测 

分 类 号：S41-33[农业科学—植物保护]