番茄褪绿病毒TaqMan荧光定量PCR快速检测方法的建立与应用

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

【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。

【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 1000 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.