Scientia Agricultura Sinica ›› 2016, Vol. 49 ›› Issue (1): 103-109.doi: 10.3864/j.issn.0578-1752.2016.01.009

• PLANT PROTECTION • Previous Articles     Next Articles

Development of a RT-LAMP Assay for Detection of Grapevine virus A

ZHANG Yong-jiang1, XIN Yan-yan1, LI Gui-fen1, QIAN Yi-ke2   

  1. 1Institute of Plant Quarantine, Chinese Academy of Inspection and Quarantine, Beijing 100176
    2Yili Entry-Exit Inspection and Quarantine Bureau, Yining 835000, Xinjiang
  • Received:2015-07-31 Online:2016-01-01 Published:2016-01-01

Abstract: 【Objective】Grapevine virus A (GVA) is one of the most important pathogens causing grapevine rugose wood complex, which is mainly transmitted by grafting using seedlings with GVA in the field. The production using free-GVA grape seedlings is the radical measure for the control of GVA disease, while the simple and sensitive detection method is the effective guarantee for the free-GVA seedlings screening. The objective of this study is to carry out the study of reverse transcription loop- mediated isothermal amplification technology (RT-LAMP), and to establish the RT-LAMP specific method for the detection of GVA.【Method】 Six specific primers for GVA detection including GVA-FIP (5′-CTTACAGCCACGCTCAGAGTCC-CGTGGGAAG TTGGTTGTGT-3′), GVA-BIP (5′-GCCCGTCAAAGGGGCTACAC-TCATAGGCGTTCTGTGCGA-3′), GVA-F3 (5′-AGAAGATG GGGATAGACCCG-3′), GVA-B3 (5′-CCGCCATTAACACGAGGAA-3′), GVA-LF (5′-ATCCTTCCCACCAGCTCGG-3′) and GVA-LB (5′-TCAGGCAGATGTGTGAACCT-3′) were designed using GVA coat protein (CP) gene sequences after a comparison analysis and design using primer design software Primer Explore 4.0. Different RT-LAMP reaction temperatures including 59, 61, 63 and 65℃ were experimented in order to determine the optimal reaction temperature according to the appearing order of the amplification curve within 1 h. The total RNAs of three other grape-infecting viruses including Grapevine rupestris stem pitting-associated virus (GRSPaV), Grapevine leafroll-associated virus (GLRaV), Grapevine fleck virus (GFKV) and the negative control (healthy grape leaf named NC) were used in this study to determine the specificity of the RT-LAMP method. The total RNA of GVA was ten-fold serially diluted including 101, 102, 103, 104, 105 and 106 dilutions and used as the templates for the sensitivity comparison between RT-LAMP and conventional RT-PCR. The RT-LAMP result could be judged by a real-time amplification curve, the curve of the positive sample appeared at the turbidity meter while no curve was observed in the negative samples. The RT-LAMP result could be also judged by a dye color reaction, the color of the positive sample was green and the color of the negative sample was orange after adding SYBR Green I in the reaction liquid.【Result】The faster and specific RT-LAMP method for the detection of GVA was developed, and the optimal reaction temperature was 65℃. The method could obtain an amplification curve only in 27 min, while the detection results could be obtained in 1.5 h using conventional RT-PCR. Specificity experiments indicated that the amplification curve could only be obtained from the total RNA of GVA and the color of the reaction liquid changed to green, which showed positive; while the other three viruses and the healthy control did not appear on the amplification curve and the color of the reaction liquids were orange, which showed negative. RT-LAMP detection results could be directly observed by the naked eye, which was easier than the results judgment of the conventional RT-PCR. Sensitivity experiments indicated that RT-LAMP could detect 101, 102, 103, 104 and 105 diluent total RNA templates of GVA, while the conventional RT-PCR could only detect 101, 102, 103 and 104 diluent total RNA templates of GVA, which showed the sensitivity of the former was 10 times higher than the sensitivity of the latter. 【Conclusion】The RT-LAMP method developed in this study could be used to detect GVA rapidly and specifically, which provided technical support for the screening of GVA-free grape seedlings and was suitable for the detection and identification of GVA in the entry quarantine, seedling breeding and field monitoring works by quarantine, research and production organizations.

Key words: Grapevine virus A, RT-LAMP, detection

[1]    杨相昆, 牛建新, 王林, 代永欣. 葡萄粗皮综合症研究进展. 北方果树, 2007(1): 1-4.
Yang X K, Niu J X, Wang L, Dai Y X. Advances in research of grapevine Rugose wood complex, RW. Northern Fruits, 2007(1): 1-4. (in Chinese)
[2]    王建辉, 刘晓, 席德慧, 袁澍, 蒋彧, 杨辉, 杜俊波, 张中伟, 陈克玲, 林宏辉. 葡萄A病毒四川分离物的外壳蛋白基因克隆与原核表达. 园艺学报, 2008, 35(7): 967-972.
Wang J H, Liu X, Xi D H, Yuan S, Jiang Y, Yang H, Du J B, Zhang Z W, Chen K L, Lin H H. Cloning and prokaryotic expression of CP gene of Grapevine virus A Sichuan isolate. Acta Horticulturae Sinica, 2008, 35(7): 967-972. (in Chinese)
[3]    魏梅生, 马洁, 李桂芬. 葡萄A病毒的IC-RT-PCR检测研究. 北方园艺, 2015(1): 99-103.
Wei M S, Ma J, Li G F. Study on detection of Grapevine virus A by immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR). Northern Horticulture, 2015(1): 99-103. (in Chinese)
[4]    Goszczynski D E, Jooste A E C. Identification of divergent variants of Grapevine virus A. European Journal of Plant Pathology, 2003, 109: 397-403.
[5]    Boscia D, Savino V, Minafra A, Namba S, Elicio V, Castellano M A, Gonsalves D, Martelli G P. Properties of a filamentous virus isolated from grapevines affected by corky bark. Archives of Virology, 1993, 130: 109-120.
[6]    Gambino G, Gribaudo I. Simultaneous detection of nine grapevine viruses by multiplex reverse transcription-polymerase chain reaction with coamplification of a plant RNA as internal control. Phytopathology, 2006, 96(11): 1223-1229.
[7]    王建辉, 刘建军, 陈克玲, 李洪雯, 席德慧, 林宏辉. 葡萄卷叶伴随3型病毒和葡萄A病毒的多重检测及其系统进化分析. 园艺学报, 2011, 38(12): 2401-2410.
Wang J H, Liu J J, Chen K L, Li H W, Xi D H, Lin H H. Optimizing multiple detection and phylogenetic studies on Grapevine leafroll associated virus-3 and Grapevine virus A.Acta Horticulturae Sinica, 2011, 38(12): 2401-2410. (in Chinese)
[8]    王建辉, 刘建军, 陈克玲, 李洪雯, 何建, 关斌. 三种葡萄病毒的RT-PCR检测和系统进化分析. 果树学报, 2013, 30(2): 197-201.
Wang J H, Liu J J, Chen K L, Li H W, He J, Guan B. RT-PCR detections and phylogenetic studies on three viruses from grapevine. Journal of Fruit Science, 2013, 30(2): 197-201. (in Chinese)
[9]    任芳, 范旭东, 董雅凤, 张尊平, 王教敏. 葡萄A病毒辽宁分离物外壳蛋白基因克隆及序列变异分析//中国植物病理学会2012年学术年会论文集. 北京: 中国农业科学技术出版社, 2012.
Ren F, Fan X D, Dong Y F, Zhang Z P, Wang J M. Cloning and prokaryotic expression of CP gene of Grapevine virus A Liaoning isolate//The Chinese Society of Plant Pathology, 2012 Academic Essays. Beijing: China Agricultural Science and Technology Press, 2012. (in Chinese)
[10]   任芳, 董雅凤, 张尊平, 范旭东, 胡国君, 朱红娟. 葡萄A病毒外壳蛋白原核表达及抗血清制备. 植物病理学报, 2014, 44(3): 327-331.
Ren F, Dong Y F, Zhang Z P, Fan X D, Hu G J, Zhu H J. Prokaryotic expression of Grapevine virus A coat protein and antiserum preparation. Acta Phytopathologica Sinica, 2014, 44(3): 327-331. (in Chinese)
[11]   范旭东, 董雅凤, 张尊平, 任芳, 李亚惠. 葡萄4种病毒多重RT-PCR检测体系的建立. 园艺学报, 2012, 39(5): 949-956.
Fan X D, Dong Y F, Zhang Z P, Ren F, Li Y H. Multiplex RT-PCR for simultaneous detection of four grapevine viruses. Acta Horticulturae Sinica, 2012, 39(5): 949-956. (in Chinese)
[12]   Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T. Loop-mediated isothermal amplification of DNA. Nucleic Acids Research, 2000, 28(12): e63.
[13]   Minafra A, Saldarelli P, Martelli G P. Grapevine virus A: nucleotide sequence, genome organization, and relationship in the Trichovirus genus. Archives of Virology, 1997, 142: 417-423.
[14]   Liu Y H, Wang Z D, Qian Y M, Mu J M, Shen L I, Wang F L. Rapid detection of Tobacco mosaic virus using the reverse transcription loop-mediated isothermal amplification method. Archives of Virology, 2010, 155: 1681-1685.
[15]   Fukuta S, Ohishi K, Yoshida K, Mizukami Y, Ishida A, Kanbe M. Development of immunocapture reverse transcription loop-mediated isothermal amplification for the detection of Tomato spotted wilt virus from chrysanthemum. Journal of Virological Methods, 2004, 121(1): 49-55.
[16]   Varga A, James D. Use of reverse transcription loop-mediated isothermal amplification for the detection of Plum pox virus. Journal of Virological Methods, 2006, 138: 184-190.
[17]   Boubourakas I N, Fukuta S, Kyriakopoulou P E. Sensitive and rapid detection of Peach latent mosaic viroid by the reverse transcription loop-mediated isothermal amplification. Journal of Virological Methods, 2009, 160: 63-68.
[18]   Tsutsumin N, Yanagisawa H, Fujiwara Y, Ohara T. Detection of Potato spindle tuber viroid by reverse transcription loop-mediated isothermal amplification. Research Bulletin of the Plant Protection Service, Japan, 2010, 46: 61-67.
[19]   陈柳, 尚巧霞, 陈笑瑜, 邢冬梅, 冉策, 魏艳敏, 赵晓燕, 刘正坪. 草莓轻型黄边病毒RT-LAMP检测方法的建立. 中国农业科学, 2015, 48(3): 613-620.
Chen L, Shang Q X, Chen X Y, Xing D M, Ran C, Wei Y M, Zhao X Y, Liu Z P. Detection of Strawberry mild yellow edge virus by RT-LAMP. Scientia Agricultura Sinica, 2015, 48(3): 613-620. (in Chinese)
[20]   闻伟刚, 杨翠云, 崔俊霞, 张颖. RT-LAMP技术检测菜豆荚斑驳病毒的研究. 植物保护, 2010, 36(6): 139-141.
Wen W G, Yang C Y, Cui J X, Zhang Y. Detection of Bean pod mottle virus by RT-LAMP. Plant Protection, 2010, 36(6): 139-141. (in Chinese)
[21]   Peng J, Zhan Y F, Zeng F Y, Long H B, Pei Y L, Guo J R. Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Fusarium oxysporum f. sp. niveum in soil. FEMS Microbiology Letters, 2013, 349(2): 127-134.
[22]   Lucchi N W, Demas A, Narayanan J, Sumari D, Kabanywanyi A, Kachur S P, Barnwell J W, Udhayakumar V. Real-time fluorescence loop mediated isothermal amplification for the diagnosis of malaria. PLoS One, 2010, 5(10): e13733.
[23]   Ge Y Y, Wu B, Qi X, Zhao K C, Guo X L, Zhu Y F, Qi Y H, Shi Z Y, Zhou M H, Wang H, Cui L B. Rapid and sensitive detection of novel avian-origin influenza A (H7N9) virus by reverse transcription loop-mediated isothermal amplification combined with a lateral-flow device. PLoS One, 2013, 8(8): e69941.
[24]   Tomita N, Mori Y, Kanda H, Notomi T. Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products. Nature Protocols, 2008, 3(5): 877-882.
[25]   刘佳, 黄丛林, 吴忠义, 张秀海, 王永勤. 环介导等温扩增技术检测菊花中番茄不孕病毒. 中国农业科学, 2010, 43(6): 1288-1294.
Liu J, Huang C L, Wu Z Y, Zhang X H, Wang Y Q. Detection of Tomato aspermy virus infecting chrysanthemums by LAMP. Scientia Agricultura Sinica, 2010, 43(6): 1288-1294. (in Chinese)
[26]   Fukuta S, Iida T, Mizukami Y, Ishida A, Ueda J, Kanbe M, Ishimoto Y. Detection of Japanese yam mosaic virus by RT-LAMP. Archives of Virology, 2003, 148: 1713-1720.
[27]   王永江, 周彦, 李中安, 苏华楠, 黄爱军, 唐科志, 周常勇. 柑橘衰退病毒RT-LAMP快速检测方法的建立. 中国农业科学, 2013, 46(3): 517-524.
Wang Y J, Zhou Y, Li Z A, Su H N, Huang A J, Tang K Z, Zhou C Y. A RT-LAMP assay for detection of Citrus tristeza virus. Scientia Agricultura Sinica, 2013, 46(3): 517-524. (in Chinese)
[28]   周彤, 杜琳琳, 范永坚, 周益军. 水稻黑条矮缩病毒RT-LAMP快速检测方法的建立. 中国农业科学, 2012, 45(7): 1285-1292.
Zhou T, Du L L, Fan Y J, Zhou Y J. Development of a RT-LAMP assay for rapid detection of Rice black-streaked dwarf virus. Scientia Agricultura Sinica, 2012, 45(7): 1285-1292. (in Chinese)
[1] DUAN Yu,XU JianJian,MA ZhiMin,BIN Yu,ZHOU ChangYong,SONG Zhen. Detection of Citrus Leaf Blotch Virus by Reverse Transcription- Recombinase Polymerase Amplification (RT-RPA) [J]. Scientia Agricultura Sinica, 2021, 54(9): 1904-1912.
[2] Xue BAI,Teng HUI,ZhenYu WANG,YunGang CAO,DeQuan ZHANG. Determination of 5 Nitropolycyclic Aromatic Hydrocarbons in Roasted Meat Products by High Performance Liquid Chromatography- Fluorescence Detection [J]. Scientia Agricultura Sinica, 2021, 54(5): 1055-1062.
[3] Tao WANG,Yu HAN,Li PAN,Bing WANG,MaoWen SUN,Yi WANG,YuZi LUO,HuaJi QIU,Yuan SUN. Development of a TaqMan Real-Time PCR Targeting the MGF360-13L Gene of African Swine Fever Virus [J]. Scientia Agricultura Sinica, 2021, 54(5): 1073-1080.
[4] JiaJia LI,HuiLong HONG,MingYue WAN,Li CHU,JingHui ZHAO,MingHua WANG,ZhiPeng XU,Yin ZHANG,ZhiPing HUANG,WenMing ZHANG,XiaoBo WANG,LiJuan QIU. Construction and Application of Detection Model for the Chemical Composition Content of Soybean Stem Based on Near Infrared Spectroscopy [J]. Scientia Agricultura Sinica, 2021, 54(5): 887-900.
[5] MA ZhiMin,XU JianJian,DUAN Yu,WANG ChunQing,SU Yue,ZHANG Qi,BIN Yu,ZHOU ChangYong,SONG Zhen. Establishment of RT-RPA for Citrus Yellow Vein Clearing Virus (CYVCV) Detection [J]. Scientia Agricultura Sinica, 2021, 54(15): 3241-3249.
[6] CHEN PengFei,MA Xiao. Research Status and Trends of Automatic Detection of Crop Planting Rows [J]. Scientia Agricultura Sinica, 2021, 54(13): 2737-2745.
[7] HUI YuanYuan,PENG HaiShuai,WANG BiNi,ZHANG FuXin,LIU YuFang,JIA Rong,REN Rong. Research Progress of Food-Borne Pathogen Detection Based on Electrochemical and Optical Aptasensors [J]. Scientia Agricultura Sinica, 2021, 54(11): 2419-2433.
[8] ZHANG QingAn,CHEN BoYu. Research Progress of Four Sulfur Compounds Related to Red Wine Flavor [J]. Scientia Agricultura Sinica, 2020, 53(5): 1029-1045.
[9] MoRan XU,RuiMing LIN,FengTao WANG,Jing FENG,ShiChang XU. Evaluation of Resistance to Stripe Rust and Genetic Diversity and Detection of Resistance Genes in 103 Wheat Cultivars (Lines) [J]. Scientia Agricultura Sinica, 2020, 53(4): 748-760.
[10] GUAN FangNian,LONG Li,YAO FangJie,WANG YuQi,JIANG QianTao,KANG HouYang,JIANG YunFeng,LI Wei,DENG Mei,LI Hao,CHEN GuoYue. Evaluation of Resistance to Stripe Rust and Molecular Detection of Important Known Yr Gene(s) of 152 Chinese Wheat Landraces from the Huang-huai-hai [J]. Scientia Agricultura Sinica, 2020, 53(18): 3629-3637.
[11] YANG HongKai,YANG JingWen,SHEN JianGuo,CAI Wei,GAO FangLuan. Multi-Gene-Based PCR Detection and Identification of Chilli veinal mottle virus [J]. Scientia Agricultura Sinica, 2020, 53(16): 3412-3420.
[12] ZHANG Ming,WANG Teng,LI Peng,DENG Lie,ZHENG YongQiang,YI ShiLai,LÜ Qiang,SUN RongRong. Surface Defect Detection of Navel Orange Based on Region Adaptive Brightness Correction Algorithm [J]. Scientia Agricultura Sinica, 2020, 53(12): 2360-2370.
[13] LI WenXue, XIAO RuiGang, LÜ MiaoMiao, DING Ning, SHI HuaRong, GU PeiWen. Establishment and Application of Real-Time PCR for Quantitatively Detecting Plasmopara viticola in Vitis vinifera [J]. Scientia Agricultura Sinica, 2019, 52(9): 1529-1540.
[14] Peng LI,Ming ZHANG,XiangSheng DAI,Teng WANG,YongQiang ZHENG,ShiLai YI,Qiang LÜ. Real-Time Estimation of Citrus Canopy Volume Based on Laser Scanner and Irregular Triangular Prism Module Method [J]. Scientia Agricultura Sinica, 2019, 52(24): 4493-4504.
[15] XueFei MAO,JiXin LIU,YongZhong QIAN. Technical Review of Fast Detection of Heavy Metals in Soil [J]. Scientia Agricultura Sinica, 2019, 52(24): 4555-4566.
Full text



[1] Qi-Feng LI. Farmers' acceptance and response to new agricultural technology based on participatory rural appraisal[J]. Scientia Agricultura Sinica, 2008, 41(7): 1963 -1968 .
[2] . Progress of Sugar Accumulation Mechanism Researches in Higher Plant Fruits[J]. Scientia Agricultura Sinica, 2008, 41(4): 1151 -1157 .
[3] . Analysis for Genetic effect and Heterosis of insect resistant transgenic cotton crosses in different ecological environments[J]. Scientia Agricultura Sinica, 2007, 40(5): 1056 -1063 .
[4] . Research on cloning and expression of VPg1-2, VPg2-3, VPg3 gene fragments of foot-and-mouth disease virus and antibody dynamics of expressed VPg1-2 protein[J]. Scientia Agricultura Sinica, 2007, 40(3): 601 -607 .
[5] . [J]. Scientia Agricultura Sinica, 2007, 40(5): 916 -924 .


Effects of Chemical Fertilizer and Organic Manure on Rice Yield and Soil Fertility

[J]. Scientia Agricultura Sinica, 2009, 42(2): 543 -551 .
[7] . [J]. Scientia Agricultura Sinica, 2008, 41(11): 3845 -3850 .
[8] . [J]. Scientia Agricultura Sinica, 1962, 03(04): 44 -47 .
[9] . [J]. Scientia Agricultura Sinica, 1993, 26(04): 71 -78 .
[10] . [J]. Scientia Agricultura Sinica, 1997, 30(06): 43 -48 .