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

【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。

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

【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.