番茄褪绿病毒TaqMan荧光定量PCR快速检测方法的建立与应用

被引：4
作者：
王吉成
李洁
丁天波
褚栋
机构：
[1] 青岛农业大学植物医学学院山东省植物病虫害综合防控重点实验室
来源：
昆虫学报 | 2020年 / 63卷 / 02期
关键词：
番茄褪绿病毒; 烟粉虱; 外壳蛋白; 携毒率; 扩增效率; TaqMan RT-qPCR;
D O I：
10.16380/j.kcxb.2020.02.005
中图分类号：
S432.41 [];
学科分类号：
090401 ;
摘要：
【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1 000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。
引用
收藏
页码：159 / 165
页数：7
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