孙勋勋,杨宏宇,周轶楠,等.桑花叶型萎缩病相关病毒的实时荧光定量PCR检测方法[J].广东农业科学,2019,(6-7):- |
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桑花叶型萎缩病相关病毒的实时荧光定量PCR检测方法 |
A real-time fluorescence quantitative PCR detection method for the Mulberry Mosaic Dwarf associated Virus |
投稿时间:2019-05-13 修订日期:2019-05-13 |
DOI: |
中文关键词: 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 |
英文关键词: Mulberry Geminivirus Mulberry mosaic dwarf disease PCR detection real-time fluorescence quantitative PCR |
基金项目:现代农业产业技术体系建设专项资金(CARS-18-ZJ0304),2017年省级农业发展和农村工作专项资金(2017LM4168) |
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中文摘要: |
【目的】 为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、陕西和江西六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,最低的有效质粒检测浓度为8 copies/μL,是普通PCR灵敏度的24倍,并且MMDaV实时荧光定量PCR检测方法对六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,可被应用于宿主体内MMDaV的定量检测。 |
英文摘要: |
【Objective】 The purpose of this study is 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 primer, construct the positive standard plasmids of MMDaV and establish the standard curve of the positive standard 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 mulberry leaf curling, mosaic and yellowing in 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 effective plasmid detection concentration of 8 copies/μL, which was 24 times as sensitive as conventional PCR. The detection method has good detection results for mulberry disease leaf samples in 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 of MMDaV has high efficiency and sensitivity. It can be applied to detect MMDaV in host plants quantitatively. |
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