Scientia Agricultura Sinica ›› 2013, Vol. 46 ›› Issue (3): 517-524.doi: 10.3864/j.issn.0578-1752.2013.03.008

• PLANT PROTECTION • Previous Articles     Next Articles

A RT-LAMP Assay for Detection of Citrus Tristeza Virus

 WANG  Yong-Jiang, ZHOU  Yan, LI  Zhong-An, SU  Hua-Nan, HUANG  Ai-Jun, TANG  Ke-Zhi, ZHOU  Chang-Yong   

  1. 1.Citrus Research Institute, Chinese Academy of Agricultural Sciences, Chongqing 400712
    2.Institute of Plant Protection, Henan Academy of Agricultural Sciences, Zhengzhou 450002
    3.Graduate School of Chinese Academy of Agricultural Sciences, Beijing 100081
  • Received:2012-07-02 Online:2013-02-01 Published:2012-10-10

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Abstract: 【Objective】 The objective of this study is to establish a quick and accurate detection technique of Citrus tristeza virus by reverse transcription loop-mediated isothermal amplification (RT-LAMP). 【Method】 According to published GenBank sequences, two pairs of primers were designed in the conserved region of CTV p25. The concentration of primers, reaction time and other conditions of RT-LAMP were optimized in order to improve specificity and sensitivity.【Result】 A rapid and specific RT-LAMP method for detection of CTV was established. Sensitivity of the RT-LAMP assay was 100-fold higher than a standard RT-PCR method. The RT-LAMP products were confirmed by digestion with Acc I restriction enzyme. Products amplified by RT-LAMP were visible to add SYBR Green I, suggesting that it can be used to detect CTV directly. 【Conclusion】The detection process can be speeded up by using RT-LAMP under routine conditions at low cost and high accuracy with simple facilities and can be used as an excellent method for CTV detection in both research institutions and rural areas.

Key words: RT-LAMP , Ctrus tristeza virus (CTV) , RT-PCR , detection

[1]Licciardello G, Raspagliesi D, Bar-Joseph M, Catara A. Characterization of isolates of Citrus tristeza virus by sequential analyses of enzyme immunoassays and capillary electrophoresis- single-strand conformation polymorphisms. Journal of Virological Methods, 2012, 181(2): 138-147.

[2]Vidal E, Yokomi R K, Moreno A, Bertolini E, Cambra M. Calculation of diagnostic parameters of advanced serological and molecular tissue-print methods for detection of Citrus tristeza virus: a model for other plant pathogens. Phytopathology, 2012, 102(1): 114-121.

[3]Albiach-Marti M R, Robertson C, Gowda S, Tatineni S, Belliure B, Garnsey S M, Folimonova S Y, Moreno P, Dawson W O. The pathogenicity determinant of Citrus tristeza virus causing the seedling yellows syndrome maps at the 3′-terminal region of the viral genome. Molecular Plant Pathology, 2010, 11(1): 55-67.

[4]Peroni L A, Lorencini M, dos Reis J R R, Machado M A, Stach-Machado D R. Differential diagnosis of Brazilian strains of Citrus tristeza virus by epitope mapping of coat protein using monoclonal antibodies. Virus Research, 2009, 145(1): 18-25.

[5]Garnsey S M, Permar T A, Cambra M, Henderson C T. Direct tissue blot immunoassay (DTBIA) for detection of Citrus tristeza virus (CTV)//Proceedings of the 12th Conference of the International Organization of Citrus Virus, 1993: 39-50.

[6]Terrada E, Kerschbaumer R J, Giunta G, Galeffi P, Himmler G, Cambra M. Fully recombinant enzyme-linked immunosorbent assays using genetically engineered single-chain antibody fusion proteins for detection of Citrus tristeza virus. Phytopathology, 2000, 90(12): 1337-1344.

[7]Roy A, Ananthakrishnan G, Hartung J S, Brlansky R H. Development and application of a multiplex reverse-transcription polymerase chain reaction assay for screening a global collection of Citrus tristeza virus isolates. Phytopathology, 2010, 100(10): 1077-1088.

[8]Nolasco G, Santos C, Silva G, Fonseca F. Development of an asymmetric PCR-ELISA typing method for citrus tristeza virus based on the coat protein gene. Journal of Virological Methods, 2009, 155(2): 97-108.

[9]Adkar-Purushothama C R, Maheshwar P K, Sano T, Janardhana G R. A sensitive and reliable RT-nested PCR assay for detection of Citrus tristeza virus from naturally infected citrus plants. Current Microbiology, 2011, 62(5): 1455-1459.

[10]Ruiz-Ruiz S, Moreno P, Guerri J, Ambros S. Discrimination between mild and severe Citrus tristeza virus isolates with a rapid and highly specific real-time reverse transcription-polymerase chain reaction method using TaqMan LNA probes. Phytopathology, 2009, 99(3): 307-315.

[11]Yokomi R K, Saponari M, Sieburth P J. Rapid differentiation and identification of potential severe strains of Citrus tristeza virus by real-time reverse transcription-polymerase chain reaction assays. Phytopathology, 2010, 100(4): 319-327.

[12]Ananthakrishnan G, Venkataprasanna T, Roy A, Brlansky R H. Characterization of the mixture of genotypes of a Citrus tristeza virus isolate by reverse transcription-quantitative real-time PCR. Journal of Virological Methods, 2010, 164(1/2): 75-82.

[13]Notomi T, Okayma H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T. Loop-mediated isothermal amplification of DNA. Nucleic Acids Research, 2000, 28(12): 63.

[14]Wastling S L, Picozzi K, Kakembo A S L, Welburn S C. LAMP for human African trypanosomiasis: a comparative study of detection formats. PLoS Neglected Tropical Disease, 2010, 4(11): e865.

[15]Wang X, Zhu J P, Zhang Q, Xu Z G, Zhang F, Zhao Z H, Zheng W Z, Zheng L S. Detection of enterovirus 71 using reverse transcription loop-mediated isothermal amplification (RT-LAMP). Journal of Virological Methods, 2012, 179(2): 330-334.

[16]Le T H, Nguyen N T B, Truong N H, Van De N. Development of mitochondrial loop-mediated isothermal amplification for detection of the small liver fluke Opisthorchis viverrini (Opisthorchiidae; Trematoda; Platyhelminthes). Journal of Clinical Microbiology, 2012, 50(4): 1178-1184.

[17]李健, 熊炜, 方雪恩, 王巧全, 姜平. 猪瘟病毒RT-LAMP检测方法的建立. 中国兽医科学, 2010, 40(10): 1033-1038.

Li J, Xiong W, Fang X E, Wang Q Q, Jiang P. Establishment of RT-LAMP system for rapid detection of classical swine fever virus. Chinese Veterinary Science, 2010, 40(10): 1033-1038. (in Chinese)

[18]Yoshida H, Sakoda Y, Endo M, Motoshima M, Yoshino F, Yamamoto N, Okamatsu M, Soejima T, Senba S, Kanda H, Kida H. Evaluation of the reverse transcription loop-mediated isothermal amplification (RT-LAMP) as a screening method for the detection of influenza viruses in the fecal materials of water birds. The Journal of Veterinary Medical Science, 2011, 73(6): 753-758.

[19]Chen L, Fan X Z, Wang Q, Xu L, Zhao Q Z, Zhou Y C, Liu J, Tang B, Zou X Q. A novel RT-LAMP assay for rapid and simple detection of classical swine fever virus. Virology Sinica, 2010, 25(1): 59-64.

[20]He Y Q, Zong W P, You Z Y, Wang X H, Yu S Y, Yang H, Dai Y C, Hu G F. Detection of human Enterovirus 71 reverse transcription loop-mediated isothermal amplification (RT-LAMP). Letters in Applied Microbiology, 2012, 54(3): 233-239.

[21]鑫婷, 侯少华, 贾红, 郭晓宇, 丁家波, 李延鹏, 丁敏, 朱鸿飞. 猪呼吸与繁殖综合症病毒RT-LAMP检测方法的建立. 中国农业科学, 2010, 43(1): 185-191.

Xin T, Hou S H, Jia H, Guo X Y, Ding J B, Li Y P, Ding M, Zhu H F. Development of a novel RT-LAMP assay for porcine reproductive and respiratory syndrome virus. Scientia Agricultura Sinica, 2010, 43(1): 185-191. (in Chinese)

[22]刘中勇, 曾小娜, 朱道中, 房红莹, 蒋启荣, 吴志强, 罗满林. 猪瘟病毒RT-LAMP快速检测方法的建立. 中国畜牧兽医, 2010, 37(6): 71-74.

Liu Z Y, Zeng X N, Zhu D Z, Fang H Y, Jiang Q R, Wu Z Q, Luo M L. Establishment of rapid reverse transcriptase loop-mediated isothermal amplification for detection of CSFV. China Animal Husbandry & Veterinary Medicine, 2010, 37(6): 71-74. (in Chinese)

[23]王永, 兰青阔, 赵新, 朱珠, 程奕. 转基因作物外源转基因成分环介导等温扩增技术检测方法的建立及应用. 中国农业科学, 2009, 42(4): 1473-1477.

Wang Y, Lan Q K, Zhao X, Zhu Z, Cheng Y. Development and application of loop-mediated isothermal amplification for detection of genetically modified crops. Scientia Agricultura Sinica, 2009, 42(4): 1473-1477. (in Chinese)

[24]刘彩霞, 梁成珠, 徐彪, 高宏伟, 林超, 孙敏. 抗草甘膦转基因大豆及加工品LAMP检测研究. 大豆科学, 2009, 28(2): 305-309.

Liu C X, Liang C Z, Xu B, Gao H W, Lin C, Sun M. Detection of genetically modified soybean and products by LAMP reaction. Soybean Science, 2009, 28(2): 305-309. (in Chinese)

[25]Chaudhary A A, Hemant, Mohsin M, Ahmad A. Application of loop-mediated isothermal amplification (LAMP)-based technology for authentication of Catharanthus roseus (L.) G. Don. Protoplasma, 2012, 249(2): 417-422.

[26]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)

[27]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)

[28]Harper S J, Ward L I, Clover G R. Development of LAMP and real-time PCR methods for the rapid detection of Xylella fastidiosa for quarantine and field applications. Phytopathology, 2010, 100(12): 1282-1288.

[29]Zhao L, Cheng J, Hao X, Tian X, Wu Y. Rapid detection of tobacco viruses by reverse transcription loop-mediated isothermal amplification. Archives of Virology, 2012, 157(12): 2291-2298.

[30]Peng J, Shi M, Xia Z, Huang J, Fan Z F. Detection of cucumber mosaic virus isolates from banana by one-step reverse transcription loop-mediated isothermal amplification. Archives of Virology, 2012, 157(11): 2213-2217.

[31]Fukuta S, Takeyama K, Suzuki M, Shichi A, Ichikawa K, Nakanishi  H. Detection of Kyuri green mottle mosaic virus from soil by the immunocapture reverse transcription loop-mediated isothermal amplification reaction. Plant Pathology Journal, 2012, 11: 51-59.

[32]Ju H J. Simple and rapid detection of potato leafroll virus by reverse transcription loop-mediated isothermal amplification. The Plant Pathology Journal, 2011, 27(4): 385-389.

[33]Lee M S, Yang M J, Hseu Y C, Lai G H, Chang W T, Hsu Y H, Lin M K. One-step reverse transcription loop-mediated isothermal amplification assay for rapid detection of Cymbidium mosaic virus. Journal of Virological Methods, 2011, 173(1): 43-48.

[34]Gillings M, Broadbent P, Indsto J, Lee R. Characterisation of isolates and strains of citrus tristeza closterovirus using restriction analysis of the coat protein gene amplified by the polymerase chain reaction. Journal of Virological Methods, 1993, 44: 305-317.

[35]易龙. 柑橘衰退病毒的进化与起源初步分析[D]. 重庆: 西南大学, 2007: 37.

Yi L. Preliminary analysis of the evolution and origin of citrus tristeza virus[D]. Chongqing: Southwest University, 2007: 37. (in Chinese)

[36]Obura E, Masiga D, Wachira F, Gurja B, Khan Z R. Detection of phytoplasma by loop-mediated isothermal amplification of DNA (LAMP). Journal of Microbiology Methods, 2011, 84(2): 312-316.

[37]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.
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