基于重组酶介导等温扩增-侧流层析试纸条的玉米褪绿斑驳病毒快速检测方法  

Rapid detection of maize chlorotic mottle virus based on recombinase-aided amplification-lateral flow assay

作　　者：邝瑞瑞 雷荣[2] 孙夕雯 赵振兴 焦志远 张永江[2] 范在丰 Kuang Ruirui;Lei Rong;Sun Xiwen;Zhao Zhenxing;Jiao Zhiyuan;Zhang Yongjiang;Fan Zaifeng(Key Laboratory of Surveillance and Management for Plant Quarantine Pests,Ministry of Agriculture and Rural Affairs,College of Plant Protection,China Agricultural University,Beijing 100193,China;Institute of Plant Quarantine,Chinese Academy of Inspection and Quarantine,Beijing 100176,China;College of Land and Environment,Shenyang Agricultural University,Shenyang 110866,Liaoning Province,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,北京100193 [2]中国检验检疫科学研究院植物检疫研究所,北京100176 [3]沈阳农业大学土地与环境学院,沈阳110866

出　　处：《植物保护学报》2022年第5期1457-1463,共7页Journal of Plant Protection

基　　金：国家重点研发计划(2021YFD1400100,2021YFD1400103);中国检验检疫科学研究院基本科研业务费项目(2020JK048);农业农村部和财政部国家现代农业产业技术体系(CARS-02)。

摘　　要：为快速和现场检测玉米褪绿斑驳病毒(maize chlorotic mottle virus,MCMV),根据MCMV外壳蛋白(coat protein,CP)基因设计引物和探针,将一步法反转录重组酶介导的等温扩增(one-step reverse transcript recombinase-aid amplification,RT-RAA)技术与侧流层析试纸条(lateral flow assay,LFA)结合建立一种快速检测MCMV的方法,对该方法的探针浓度、反应温度和反应时间进行优化,并对优化后方法的特异性和灵敏度进行检测。结果表明,该检测方法的最佳探针浓度为0.02μmol/L,最佳反应温度为37℃,最佳反应时间为8 min;该检测方法的特异性强且对MCMV RNA的检测灵敏度为8.6×10^(-6) ng/μL,比常规RT-PCR方法的灵敏度提高10倍,对MCMV CP质粒DNA拷贝数的检测灵敏度为92.5 copies/μL,比常规RT-PCR方法的灵敏度提高10倍;该检测方法用时短,约13 min,其中RT-RAA仅需8 min,LFA仅需5 min,且操作简单,可用于MCMV的实时监测及病害的田间快速诊断。Primers and probes were designed according to the coat protein(CP)gene of maize chlorotic mottle virus(MCMV),and one-step reverse transcript recombinase-aid amplification(RT-RAA)was combined with lateral flow assay(LFA)to establish a rapid detection method for MCMV in this study.The probe concentration,reaction temperature and reaction time of the method were optimized,and the specificity and sensitivity of the optimized method were examined.The results showed that the optimal probe concentration of this method was 0.02μmol/L,the optimal reaction temperature was 37℃,and the optimal reaction time was 8 min.The detection method was highly specific,and had a detection sensitivity of 8.6×10^(-6)ng/μL for MCMV total RNA,which was ten times higher than the sensitivity of conventional RT-PCR method,and 92.5 copies/μL for MCMV CP plasmid DNA copy number,ten times higher than that of conventional RT-PCR method.The detection method is quick,takes about 13 min,among which RT-RAA needs eight min,LFA needs five min,and the operation is simple,which can be used for real-time monitoring of MCMV and rapid field diagnosis of the viral disease.

关 键 词：玉米褪绿斑驳病毒 等温扩增 侧流层析试纸条 外壳蛋白 

分 类 号：S41-30[农业科学—植物保护]