番茄褪绿病毒TaqMan荧光定量PCR快速检测方法的建立与应用

【目的】本研究旨在建立TaqMan实时荧光定量PCR(TaqMan RT-qPCR)技术,快速检测单头烟粉虱Bemisia tabaci体内的番茄褪绿病毒(tomato chlorosis virus,ToCV)。【方法】根据ToCV外壳蛋白保守序列设计了1对特异性引物和1条TaqMan探针,建立了TaqMan RT-qPCR方法;与常规PCR检测进行比较,检测该方法的灵敏度与特异性;并应用该方法对单头烟粉虱成虫体内ToCV进行了快速检测。【结果】本研究构建的TaqMan RT-qPCR检测ToCV的标准曲线,其循环阈值(Ct值)与模板浓度具有良好的线性关系,扩增效率为98%。该方法对ToCV的最低检测浓度为8.3×10 copies/μL,灵敏度是常规RT-PCR的1000倍。该方法与田间番茄两种重要病毒番茄黄化曲叶病毒(tomato yellow leaf curl virus,TYLCV)和番茄斑萎病毒(tomato spotted wilt virus,TSWV)检测无交叉反应。单头烟粉虱成虫ToCV检测结果表明,温室内ToCV侵染植株上烟粉虱携毒率为100%,田间烟粉虱的携毒率为30%。【结论】本研究建立的TaqMan RT-qPCR检测方法,可快速有效检测单头烟粉虱体内ToCV携毒情况,为该病毒病的防控提供了技术支撑。

Development and application of a TaqMan real-time fluorescent quantitative PCR method for rapid detection ofTomato chlorosis virus

【Aim】 The aim of this study is to develop a TaqMan real-time fluorescent quantitative PCR (TaqMan RT-qPCR) method to rapidly detect the tomato chlorosis virus (ToCV) in a single whitefly (Bemisia tabaci) vector. 【Methods】 A pair of specific primers and one TaqMan probe were designed based on the conserved sequence of ToCV coat protein, and then TaqMan RT-qPCR was developed for viral detection. The sensitivity and specificity of TaqMan RT-qPCR were compared to those of the conventional PCR. Finally, this method was applied to rapidly detect ToCV in a single adult of B. tabaci. 【Results】 The cycle threshold (Ct) on the standard curve of TaqMan RT-qPCR of ToCV showed a linear relationship with the template concentration, and the amplification efficiency was 98%. The minimum concentration of this virus detection method was 8.3×10 copies/μL, and the sensitivity was 1 000 times as high as that of the conventional PCR. This method had no cross-reactivity with two important tomato viruses in the field, i.e., tomato yellow leaf curl virus (TYLCV) and tomato spotted wilt virus (TSWV). Detection of ToCV in a single adult of B. tabaci showed that the viruliferous rate of ToCV in B. tabaci in greenhouses was 100% and that in the field was 30%. 【Conclusion】 The TaqMan RT-qPCR method developed in this study can detect ToCV in a single whitefly (B. tabaci) rapidly and efficiently, providing technical support for the prevention and control of this virus disease.