甘薯卷叶病毒LAMP快速检测技术的建立及应用  

Establishment and Application of a LAMP Method for the Rapid Detection of Sweet potato leaf curl virus

作　　者：李华伟[1,2] 刘中华 张鸿[1,2] 许泳清 李国良[1,2] 林赵淼 许国春[1,2] 邱永祥 纪荣昌[1,2] 罗文彬 汤浩[1,2] 邱思鑫 LI Hua-Wei;LIU Zhong-Hua;ZHANG Hong;XU Yong-Qing;LI Guo-Liang;LIN Zhao-Miao;XU Guo-Chun;QIU Yong-Xiang;JI Rong-Cang;LUO Wen-Bin;TANG Hao;QIU Si-Xin(Crop Institute Research Centre, Fujian Academy of Agricultural Sciences, Fuzhou 350013, China;Scientific Observing and Experimental Station of Tuber and Root Crops in South China, Ministry of Agriculture, Fuzhou 350013, China)

机构地区：[1]福建省农业科学院作物研究所,福州350013 [2]农业部南方薯类科学观测实验站,福州350013

出　　处：《农业生物技术学报》2019年第6期1133-1140,共8页Journal of Agricultural Biotechnology

基　　金：国家现代农业产业技术体系(CARS-10-B14);福建省科技重大专项-薯类(甘薯,马铃薯)新品种选育及安全高效栽培技术研究(No.2017NZ0002-2);福建省种业创新与产业化工程项目(No.fjzycxny2017005);福建省农业科学院创新团队项目(No.STIT2017-2-3)

摘　　要：甘薯卷叶病毒(Sweet potato leaf curl virus, SPLCV)是甘薯(Ipomoea batatas)生产上重要的病毒病之一,建立SPLCV快速检测方法,对该病的诊断及技术防控至关重要。本研究以甘薯卷叶病毒基因组的外壳蛋白基因(coat protein, cp)为靶序列,设计了一组环介导恒温核酸扩增(loop-mediated isothermal amplification, LAMP)特异性引物,以SYBR Green Ⅰ作检测指示剂,通过优化反应条件,建立了一种甘薯卷叶病毒的LAMP可视化快速检测方法,该方法在65 ℃的恒温条件下反应1 h完成检测。特异性检测结果表明,只有感染SPLCV的样品在指示剂的作用下呈现绿色,且扩增产物用2.0%琼脂糖凝胶电泳出现梯形条带,而与感染其他甘薯DNA病毒、番茄黄花曲叶病毒(Tomato yellow mosaic leaf curl virus, TYLCV)、烟草曲叶病毒(Tobacco leaf curl virus, TLCV) 3种DNA病毒均无交叉反应,表明该方法具有较高的特异性;灵敏度检测结果表明,该方法的最低检测限为1 pg/μL基因组DNA,是普通PCR的10倍,说明该方法灵敏度较高。因此本研究建立的甘薯卷叶病毒环介导等温扩增技术具有扩增快速、特异性高、简便、不需要特殊仪器,肉眼可视化观察结果,适合田间甘薯卷叶病毒样品及基层部门的快速检测。Sweet potato leaf curl virus (SPLCV), is one of the most important diseases on sweet potato (Ipomoea batatas), that infection reduced the yields of the most sweet potato growing area. Rapid and accurate detection of SPLCV is essential for diagnosis, prevention and control of the disease. In this study, a set of primers for loop-mediated isothermal amplification (LAMP) detection was designed based on the coat protein gene (cp) selected from the sequencing results of SPLCV,design a set of LAMP specific primers, with SYBR Green Ⅰ for evaluation indicator, optimize the reaction conditions, a rapid and visualized LAMP method for detection of SPLCV was established, this method were completed under isothermal conditions at 65 ℃ for 1 h. Specificity test results showed that SPLCV could be specifically detected by this method, and there was no cross reaction with other DNA pathogens such as other sweet potato DNA virus, Tomato yellow mosaic leaf curl virus (TYLCV) and Tobacco leaf curl virus (TLCV), indicated that the method had good specificity. The sensitivity results showed that the minimum detection limit of this method was 1 pg/μL of the SPLCV genomic DNA, which was 10 times of ordinary PCR. It showed that this method had high sensitivity and good repeatability. The technique of LAMP of SPLCV established in this study had the advantages of rapid amplification, high efficiency, good specificity, simple operation, no need for special instruments, visual observation results with the naked eye, and is suitable for rapid detection of SPLCV samples in the field and grass-roots departments.

关 键 词：甘薯 甘薯卷叶病毒(SPLCV) 环介导等温扩增技术(LAMP) SYBR Green Ⅰ 

分 类 号：S41-30[农业科学—植物保护]