Scientia Agricultura Sinica ›› 2013, Vol. 46 ›› Issue (3): 525-533.doi: 10.3864/j.issn.0578-1752.2013.03.009

• PLANT PROTECTION • Previous Articles     Next Articles

Development and Application of a Real-Time RT-PCR Approach for Quantification of CTV in Toxoptera citricida

 LI  Ling-Di, ZHOU  Chang-Yong, LI  Zhong-An, TIAN  Xiao, WANG  Yong-Jiang, TANG  Ke-Zhi, ZHOU  Yan, LIU  Jin-Xiang   

  1. 1.College of Horticulture and Landscape Architecture, Southwest University, Chongqing 400715
    2.Citrus Research Institute,  Chinese Academy of Agricultural Sciences, Chongqing 400712
  • Received:2012-07-30 Online:2013-02-01 Published:2012-11-06

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Abstract: 【Objective】The objective of this study is to develop a SYBR Green I real-time RT-PCR assay to detect the Citrus tristeza virus in Toxoptera citricida (Kirkaldy). 【Method】A pair of primers HD-F/R were designed within highly conservative region of CP25, and the SYBR Green I real-time RT-PCR detection system was established with optimized reaction condition. Analytical sensitivity and reproducibility were evaluated, respectively. Finally, the method was used to quantify CTV in single T. citricida. 【Result】The assay had a detection limit of 9.0 copies/μL and the sensitivity was 100 times higher than the conventional RT-PCR. The standard curve established by cRNA showed a fine linear relationship between threshold cycle and template concentration. The correlation coefficient of the standard curve was 0.998 and amplification efficiency was 104.7%. The variation coefficient of Ct value of diluted standard cRNA was less than 3.24%, indicating a good reproducibility. After 24 h acquisition access period, the estimate number of CTV targets in single T. citricida ranged from 2.5×103 to 1.24×106 copies. 【Conclusion】 The quantitative method was used for accurate determination of Citrus tristeza virus in T. citricida and could be a potential tool for studying the aphids-CTV-host interaction and CTV epidemiology.

Key words: Toxoptera citricida , Citrus tristeza virus (CTV) , real-time RT-PCR

[1]林尤剑, 谢联辉, Powell C A. 橘蚜传播柑橘衰退病毒的研究进展. 福建农业大学学报, 2001, 30(1): 59-66.

Lin Y J, Xie L H, Powell C A. Advance in Citrus tristeza virus transmission by brown citrus aphid. Journal of Fujian Agricultural University, 2001, 30(1): 59-66. (in Chinese)

[2]余冬冬, 刘永清, 王国平. 柑橘衰退病毒研究进展. 果树学报, 2003, 20(3): 224-229.

Yu D D, Liu Y Q, Wang G P. Advance in research of Citrus    tristeza virus. Journal of Fruit Science, 2003, 20(3): 224-229. (in Chinese)

[3]Rocha-Pena M A, Lee R F, Lastra R, Niblett C L, Ochoa-Corona F M, Garnsey S M, Yokomi R K. Citrus tristeza virus and its aphid vector Toxoptera citricida: threats to citrus production in the carribean and central and North America. Plant Disease, 1995, 79(5): 437-445.

[4]Carlebach R, Raccah B, Loebesntein G. Detection of potato virus Y in the aphid Myzus persicae by enzyme-linked immunosorbent assay (ELISA). Annals of Applied Biology, 1982, 101: 511-516.

[5]Mehta P, Brlansky R H, Gowda S, Yokomi R K. Reverse-transcription polymerase chain reaction detection of Citrus tristeza virus in aphids. Plant Disease, 1997, 81(9): 1066-1069. 

[6]Cambra M, Gorris M T, Olmos A, Martínez M C, Román M P, Bertolini E, López A, Carbonell E A. European diagnostic protocols (DIAGPRO) for Citrus tristeza virus in adult trees//Fifteenth International Organization Citrus Virologists Conference, California, America, 2002: 69-77. 

[7]Saponari M, Manjunath K, Yokomi R K. Quantitative detection of Citrus tristeza virus in citrus and aphids by real-time reverse transcription-PCR (TaqMan®). Journal of Virological Methods, 2008, 147: 43-53.

[8]Bertolini E, Moreno A, Capote N, Olmos A, de Luis A, Vidal E, Pérez-Panadés J, Cambra M. Quantitative detection of Citrus tristeza virus in plant tissues and single aphids by real-time RT-PCR. European Journal of Plant Pathology, 2008, 20: 177-188.

[9]Ruiz-Ruiz S, Moreno P, Guerri J, Ambrós S. A real-time RT-PCR assay for detection and absolute quantitation of Citru stristeza virus in different plant tissues. Journal of Virological Methods, 2007, 145(2): 96-105.

[10]Fronhoffs S, Totzke G, Stier S, Wernert N, Rothe M, Brüning T, Koch B, Sachinidis A, Vetter H, Ko Y. A method for the rapid construction of cRNA standard curves in quantitative real-time reverse transcription polymerase chain reaction. Molecular and Cellular Probes, 2002, 16: 99-110.

[11]Caelers A, Berishvili G, Meli M L, Eppler E, Reinecke M. Establishment of a real-time RT-PCR for the determination of absolute amounts of IGF-I and IGF-II gene expression in live and extrahepatic sites of the tilapiar. General and Comparative Endocrinology, 2004, 137: 196-204.

[12]蔡刚, 李闻捷, 丁健华, 付传刚, 沈茜. 构建cRNA作为标准曲线的实时RT-PCR检测方法. 临床检验杂志, 2004, 22(3): 172-174.

Cai G, Li W J, Ding J H, Fu C G, Shen Q. A method for the rapid construction of cRNA standard curves in real-time RT-PCR. Chinese Journal of Clinical Laboratory Science, 2004, 22(3): 172-174. (in Chinese)

[13]Vijgen L, Keyaerts E, Moës E, Maes P, Duson G, Van Ranst M. Development of one-step, real-time, quantitative reverse transcriptase PCR assays for absolute quantitation of human coronaviruses OC43 and 229E. Journal of Clinical Microbiology, 2005, 43(11): 5452-5456.

[14]Olmos A, Bertolini E, Gil M, Cambra M. Real-time assay for quantitative detection of non persistently transmitted Plum pox virus RNA targets in a single aphids. Journal of Virological Methods, 2005, 128: 151-155.

[15]黄竺筠, 张冬雷, 施建, 崔之础. RNA荧光定量标准品制备新方法的探讨. 现代检验医学杂志, 2005 , 20(6): 18-21.

Huang Z J, Zhang D L, Shi J, Cui Z C. Preparation of RNA standards for Real-time fluorescence quantitative PCR. Journal of Modern Laboratory Medicine, 2005, 20(6): 18-21. (in Chinese)

[16]朵建英, 王卫, 从喆, 刘强, 魏强. SYBR GreenⅠ实时荧光定量RT-PCR测定肠道病毒71型 (EV71) RNA拷贝数方法的建立. 中国比较医学杂志, 2010, 20(7): 27-31.

Duo J Y, Wang W, Cong Z, Liu Q, Wei Q. Quantification of EV71 viral load using real-time quantitative RT-PCR with SYBR Green I. Chinese Journal of Comparative Medicine, 2010, 20(7): 27-31. (in Chinese)

[17]邹勤. 应用real time RT-PCR研究柑橘衰退病毒弱毒株对强毒株的拮抗作用[D]. 重庆: 西南大学, 2010.

Zou Q. Cross protection of a Citrus tristeza virus mild strain against a severe strain detected by Real-time RT-PCR[D]. Chongqing: Southwest University, 2010. (in Chinese)

[18]于庆涛. 检测CTV的实时荧光RT-qPCR技术研发与应用[D]. 重庆: 西南大学, 2008.

Yu Q T. Development and application of the detection technique of Real time fluorescence RT-qPCR on Citrus tristeza virus[D]. Chongqing: Southwest University, 2008. (in Chinese)

[19]高雅红, 王进忠, 李明福, 李桂芬. 李痘病毒实时荧光定量RT-PCR检测方法的建立. 生物技术通讯, 2011, 22(1): 77-80.

Gao Y H, Wang J Z, Li M F, Li G F. Development of a real-time fluorescence quantitative RT-PCR method for the detection of   Plum pox virus. Letters in Biotechnology, 2011, 22(1): 77-80. (in Chinese)

[20]刘红光, 王中康, 曹月青, 夏玉先, 殷幼平. 应用常规RT-PCR和荧光定量RT-PCR检测柑橘衰退病毒. 植物病理学报, 2008, 38(1): 24-30.

Liu H G, Wang Z K, Cao Y Q, Xia Y X, Yin Y P. Detection of Citrus tristeza virus using conventional and fluorescence quantitative RT-PCR assays. Acta Phytopathologica Sinica, 2008, 38(1): 24-30. (in Chinese)

[21]Zhou Y, Zhou C Y, Wang X E, Liu Y Q, Liu K H, Zou Q, Xiang Y, Li Z A. Influence of the quantity and variability of Citrus tristeza virus on transmissibility by single Toxoptera citricida. Journal of Plant Pathology, 2011, 93(1): 97-103.

[22]Hashimoto Y, Valles S M, Strong C A. Detection and quantitation of Solenopsis invicta virus in ?re ants by real-time PCR. Journal of Virological Methods, 2007, 140: 132-139.

[23]Bar-Joseph M, Marcus R, Lee R F. The continuous challenge of Citrus tristeza virus control. Annual Review of Phytopathology, 1989, 27: 291-316.

[24]Raccah B, Loebenstein G, Singer S. Aphid-transmissibility variants of citrus tristeza virus in infected citrus trees. Phytopathology, 1980, 70(2): 89-93.

[25]Yokomi R K, Lastra R, Stoetzel M B, Damsteegt V D, Lee R F, Garnsey S M, Gottwald T R, Rocha-Peña M A, Niblett C L. Establishment of the brown citrus aphid (Homoptera: Aphididae) in central America and the Caribbean Basin and transmission of Citrus tristeza virus. Journal of Economic Entomology, 1994, 87(4): 1078-1085.

[26]Moreno A, Fereres A, Camber M. Quantitative estimation of plum pox virus targets acquired and transmitted by a single Myzus persicae. Archives of Virology, 2009, 154: 1391-1399.

[27]田彩娟, 张鹏波, 韩日畴. 红火蚁病毒SINV的研究进展. 中国生物防治, 2008, 24(增刊): 118-126.

Tian C J, Zhang P B, Han R C. Research progress on the Solenopsis invicta virus. Chinese Journal of Biological Control, 2008, 24(Suppl.): 118-126. (in Chinese)
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