桑花叶型萎缩病相关病毒的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 the Mulberry Mosaic Dwarf associated Virus

作　　者：孙勋勋 杨宏宇 周轶楠 刘吉平[1] SUN Xunxun;YANG Hongyu;ZHOU Yinan;LIU Jiping(College of Animal Science,South China Agricultural University,Guangzhou 510642,China)

机构地区：[1]华南农业大学动物科学学院

出　　处：《广东农业科学》2019年第8期111-117,共7页Guangdong Agricultural Sciences

基　　金：国家现代农业产业技术体系建设专项资金(CARS-18-ZJ0304);广东省农业发展和农村工作专项资金(2017LM4168)

摘　　要：【目的】建立桑花叶型萎缩病相关病毒(Mulberry Mosaic Dwarf associated Virus,MMDaV)的实时荧光定量PCR检测方法。【方法】以MMDaV保守的外壳蛋白基因为靶基因,设计特异引物,构建外壳蛋白基因序列的阳性质粒,建立阳性质粒的标准曲线,并对MMDaV特异引物的灵敏性和特异性进行检测,并使用建立的MMDaV实时荧光定量PCR检测方法检测广东、广西、海南、重庆、陕西和江西6个省区的桑病叶样品。【结果】构建的标准曲线具有良好的扩增效率(97.88%),设计的特异引物可特异的检测到MMDaV,最低的质粒检测浓度为8 copies/μL,是普通PCR灵敏度的24倍,并且MMDaV实时荧光定量PCR检测方法对6个省区的桑病叶样品均有良好的检测结果,检测的Cq值在9.69~26.17之间。【结论】建立的MMDaV实时荧光定量PCR检测方法具有高效率、特异性好和灵敏度高等特性,可被应用于寄主体内MMDaV的定量检测。【Objective】The purpose of this study was to establish a real-time fluorescence quantitative PCR detection method for the Mulberry Mosaic Dwarf associated Virus(MMDaV).【Method】As target gene,the conservative coat protein gene of MMDaV was applied to design the specific primers,construct the positive plasmids of MMDaV and establish the standard curve of the positive plasmids.The sensitivity and specificity of the particular primer of MMDaV was detected.The real-time fluorescence quantitative PCR detection method of MMDaV was applied to detect MMDaV of diseased mulberry leaf from six provinces of Guangdong,Guangxi,Hainan,Chongqing,Shaanxi,and Jiangxi.【Results】The standard curve constructed has excellent amplification efficiency(97.88%).The specific primer can accurately detect the MMDaV.The detection method has the lowest plasmid detection concentration of 8 copies/μL,the sensitivity of which was 24 times higher than that of conventional PCR.The method has good detection results for diseased mulberry leaf samples from six provinces,and the Cq value of detection is between 9.69 and 26.17.【Conclusion】The established real-time fluorescence quantitative PCR detection method for MMDaV,with high efficiency,specificity and sensitivity,can be applied to detect MMDaV in host plants quantitatively.

关 键 词：桑树 双生病毒 桑花叶型萎缩病 桑花叶型萎缩病相关病毒 实时荧光定量PCR 

分 类 号：S432.41[农业科学—植物病理学]