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.