云南烟草曲叶病毒LAMP快速检测体系的建立及初步应用  

Development and Preliminary Application of LAMP Assay for Rapid Detection of Tobacco leaf curl Yunnan virus(TbLCYnV)

作　　者：赵正婷 盖晓彤 张俊蕾 夏振远[1] 刘弟 刘雅婷[2,3,4] 姜宁 ZHAO Zheng-Ting;GE Xiao-Tong;ZHANG Jun-Lei;XIA Zhen-Yuan;LIU Di;LIU Ya-Ting;JIANG Ning(Yunnan Academy of Tobacco Agricultural Sciences,Kunming 650021,China;College of Agronomy and Biotechnology,Yunnan Agricultural University,Kunming 650201,China;College of Plant Protection,Yunnan Agricultural University,Kunming 650201,China;College of Tobacco Science,Yunnan Agricultural University,Kunming 650201,China)

机构地区：[1]云南省烟草农业科学研究院,昆明650021 [2]云南农业大学农学与生物技术学院,昆明650201 [3]云南农业大学植物保护学院,昆明650201 [4]云南农业大学烟草学院,昆明650201

出　　处：《农业生物技术学报》2024年第8期1949-1958,共10页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(32260681);云南省烟草公司科技计划项目(2021530000242031);云南省科技厅基础研究专项(202101AU070129)。

摘　　要：云南烟草曲叶病毒(Tobacco leaf curl Yunnan virus,TbLCYnV)是在中国云南保山烟草(Nicotiana tabacum)上发现的菜豆金色花叶病毒属新病毒,是云南烟草曲叶病的重要病原之一。为快速检测TbLCYnV,本研究根据TbLCYnV的外壳蛋白(coat protein,CP)基因保守核苷酸序列,设计了5组引物进行筛选,并采用单一变量法对环介导等温扩增技术(loop-mediated isothermal amplification,LAMP)反应温度、时间,体系中甜菜碱、dNTPs、Mg2+的体积摩尔浓度、内外引物比等进行逐一优化,同时与传统PCR平行对比以验证LAMP的特异性和灵敏度,应用田间采集样品验证LAMP体系的实用性,确立了Tb LCYnV的LAMP检测最佳体系。结果表明,最佳引物组为TbL-5,最适反应温度为60℃,甜菜碱、d NTPs、Mg2+的最佳终浓度分别为1.0、0.4和4.0 mmol/L,最佳内外引物浓度比为4∶1,最佳反应时间60 min。检测结果显示优化后的LAMP经SYBR GreenⅠ染色可通过肉眼直接判断结果,具有高度特异性,且灵敏度是常规PCR的10000倍。本研究为TbLCYnV的检测提供了一种便捷、高效、可靠的方法,对于Tb LCYnV的早期快速诊断和防控具有重要意义。Tobacco leaf curl Yunnan virus(TbLCYnV)is a new virus of the Begomovirus discovered on tobacco(Nicotiana tabacum)in Baoshan,Yunnan,China.It is one of the important pathogens of tobacco leaf curl disease in Yunnan.In order to quickly detect the TbLCYnV,five sets of primers were designed and screened according to the conserved nucleotide sequence of the coat protein(CP)gene of TbLCYnV,and the loop mediated isothermal amplification(LAMP)system reaction temperature,time,betaine concentration,dNTP concentration,Mg2+concentration,and concentration ratio of internal and external primers were optimized by single variable method,At the same time,parallel comparisons with traditional PCR were conducted to verify the specificity and sensitivity of LAMP.The practicality of the LAMP system was verified using field collected samples,and the optimal LAMP detection system for TbLCYnV was established.The results showed that the optimal parameters were as follows:Primer group was TbL-5,the reaction temperature was 60℃,and the final concentrations of Betaine,dNTPs,and Mg2+were 1.0,0.4,4.0 mmol/L,respectively;The optimal concentration ratio of internal and external primers was 4∶1,and the optimal reaction time was 60 min.The test results show that the optimized LAMP can be directly judged by naked eyes after staining with SYBR GreenⅠ,with high specificity and 10000 times higher sensitivity than that of conventional PCR.This study provides a convenient,efficient,and reliable method for the detection of TbLCYnV,which is of great significance for the early and rapid diagnosis and prevention and control of TbLCYnV.

关 键 词：云南烟草曲叶病毒(TbLCYnV) 外壳蛋白(CP)基因 环介导等温扩增(LAMP) 特异性 灵敏度 

分 类 号：S435.72[农业科学—农业昆虫与害虫防治]