木尔坦棉花曲叶病毒SYBR Green I实时荧光定量PCR检测方法

赵蕊; 吕利华; 陈婷; 何自福; 何余容

doi:10.7671/j.issn.1001-411X.2015.06.014

木尔坦棉花曲叶病毒SYBR Green I实时荧光定量PCR检测方法

A SYBR Green I real-time fluorescence quantitative PCR detection method for Cotton leaf curl virus

摘要

摘要:
目的木尔坦棉花曲叶病毒Cotton Leaf Curl Multan Virus（CLCuMV）是为害锦葵科植物的重要双生病毒之一，本研究的目的是建立CLCuMV定量检测方法，为进一步研究其在寄主植物内的增殖动态、在介体昆虫内循环和其与寄主、介体间互作机制提供技术手段.
方法以纯化的含CLCuMV基因组的质粒为模板，利用SYBR Green I荧光定量PCR对不同浓度的质粒样品进行CLCuMV扩增，制作标准曲线.依照建立的标准曲线，计算烟粉虱Bemisia tabaci及朱槿Hibiscus rosa-sinensis中CLCuMV的含量.
结果和结论 该定量检测法可检测到低浓度（5.2×10¹ μL^-1)的CLCuMV，其灵敏度是常规PCR的10倍，可被用于在寄主植物和介体昆虫体内CLCuMV的定量检测.

Abstract:
Objective Cotton Leaf Curl Multan Virus (CLCuMV) is one of the most important viralpathogens of Malvaceae plants. The purpose of this study is to establish an accurate detection method forCLCuMV, which can provide a technological means to study propagation dynamics of viruse in hostplants, virus generation cycle inside vector insect, and interaction mechanisms among virus, plant andvector.
Method The SYBR Green I real-time fluorescence quantitative PCR was adopted usingCLCuMV-plasmid as template and the standard curve was established. In accordance with the standardcurve, the CLCuMV contents in leaves of Hibiscus rose-sinensis and vector bodies of Benisia tabaci werecalculated.
Result and conclusion The detection sensitivity was as low as 5.2 × 10¹ μL^-1 of CLCuMV, which was 10 times as sensitive as conventional PCR. It can be applied to quantitatively detect CLCuMVin host plants and vector insects.

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