基于RPA-CRISPR/Cas12a的番茄花叶病毒可视化检测方法的建立  

Establishment of RPA-CRISPR/Cas12a-based visual detection of tomato mosaic virus

作　　者：董铮 赵振兴 范奇璇 王思元 周涛 张永江[1] DONG Zheng;ZHAO Zhenxing;FAN Qixuan;WANG Siyuan;ZHOU Tao;ZHANG Yongjiang(Chinese Academy of Inspection and Quarantine,Beijing 100176,China;College of Plant Protection,China Agricultural University,Beijing 100193,China)

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,北京100193

出　　处：《植物保护》2024年第4期235-241,共7页Plant Protection

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

摘　　要：番茄花叶病毒(tomato mosaic virus,ToMV)属于植物杆状病毒科Virgaviridae烟草花叶病毒属Tobamovirus成员,主要危害番茄和辣椒,多数茄科植物易感染,严重影响果实的品质和产量。本研究根据ToMV编码的外壳蛋白的基因保守序列,设计重组酶聚合酶扩增技术(recombinase polymerase amplification,RPA)特异性引物和簇状规则间隔短链重复序列(clustered regularly interspaced short palindromic repeats,CRISPR)及相关蛋白12a(CRISPR-associated 12a,Cas12a)的crRNA并挑选报告基因。通过优化反应体系建立了ToMV的快速可视化检测方法,当荧光报告基因FQ终浓度为400 nmol/L、Cas12a/crRNA比例为1∶5、终浓度为200 nmol·L^(-1)/1000 nmol·L^(-1)时检测信号最强,只需RPA和CRISPR/Cas12a分别反应15 min,即可在便携式蓝光照射设备下直接观察到阳性信号。该方法可特异性检测ToMV,对携带ToMV样品的RNA检测灵敏度可以达到172 ag/μL,是普通RT-PCR检测灵敏度的10000倍,可用于ToMV快速灵敏的可视化检测。Tomato mosaic virus(ToMV)is a member of the genus Tobamovirus in the family Virgaviridae.ToMV mainly infects tomato,pepper,and most Solanaceae plants,resulting in severe losses in fruit quality and yield.In this study,the specific primers of recombinase polymerase amplification(RPA)and the crRNA of clustered regularly interspaced short palindromic repeats/CRISPR-associated 12a(CRISPR/Cas12a)were designed based on the conserved sequence of the coat protein sequence of ToMV,and the reporter gene was selected.By optimizing the reaction system,a rapid visual detection method for ToMV detection was established,and the detection signal was strongest when the final concentration of fluorescent reporter FQ was 400 nmol/L,the Cas12a/crRNA ratio was 1∶5,and the final concentration was 200 nmol·L^(-1)/1000 nmol·L^(-1).With only 15 min of RPA and CRISPR/Cas12a reaction,respectively,positive signals can be directly observed under a portable blue light irradiation equipment.This method can be used for specific detection of ToMV,and the minimal detection limit in detecting the total RNA of ToMV containing samples is 172 ag/μL,10000 times that of RT-PCR-based ToMV detection.Hence,the established RPA-CRISPR/Cas12a-based detection is a rapid,sensitive and visual method for ToMV detection.

关 键 词：番茄花叶病毒 RPA CRISPR/Cas12a 可视化检测 

分 类 号：S436.412[农业科学—农业昆虫与害虫防治]