褐色橘蚜中柑橘衰退病毒实时荧光定量RT-PCR检测方法的建立与应用  

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

作　　者：李玲娣[1,2] 周常勇[2] 李中安[2] 田晓[1,2] 王永江[2] 唐科志[2] 周彦[2] 刘金香[2] 

机构地区：[1]西南大学园艺园林学院,重庆400715 [2]中国农业科学院柑桔研究所,重庆400712

出　　处：《中国农业科学》2013年第3期525-533,共9页Scientia Agricultura Sinica

基　　金：国家公益性行业(农业)科研专项(201203076-01);教育部创新团队(IRT0976);国家自然科学基金项目(30600419);西南大学基本科研业务费(XDJK2009C133);重庆市自然科学基金项目(CSTC2012JJA80036)

摘　　要：【目的】建立SYBR Green I实时荧光定量RT-PCR方法,测定褐色橘蚜(Toxoptera citricida)中柑橘衰退病毒(Citrus tristeza virus,CTV)含量。【方法】根据CTV CP25保守序列,设计特异性引物HD-F/R,通过优化得到最佳反应条件建立橘蚜中CTV实时荧光定量RT-PCR,并进行灵敏性、重复性检验,评价该方法的可行性。应用该方法测定单头橘蚜中CTV含量。【结果】该实时荧光定量RT-PCR方法最低检测限为9.0拷贝/μL,其灵敏度是常规RT-PCR的100倍。标准曲线循环阈值与模板浓度呈良好的线性关系,相关性系数为0.998,扩增效率达104.7%。批内和批间变异系数均小于3.24%,表明该方法重现性好。获毒24 h单头橘蚜中CTV最低含量为2.5×103拷贝,最高含量为1.24×106拷贝。【结论】本研究建立的实时荧光定量RT-PCR检测方法能够准确检测褐色橘蚜中的CTV,可用于研究褐色橘蚜-CTV-寄主互作关系及CTV的流行。[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/gL 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 2.5×10^3 to 1.24×10^6 copies. [Conclusion] The quantitative method was used for accurate determination of Citrus tristeza virus in T. eitricida and could be a potential tool for studying the aphids-CTV-host interaction and CTV epidemiology.

关 键 词：褐色橘蚜 柑橘衰退病毒 实时荧光定量RT-PCR 

分 类 号：S436.66[农业科学—农业昆虫与害虫防治]