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| 烟草环斑病毒的定量检测技术研究 | |
| 引用本文： | 刘梅,黄新,马占鸿,陈洪俊,李明福.烟草环斑病毒的定量检测技术研究[J].生物技术通讯,2010,21(1):73-76. |
| 作者姓名： | 刘梅 黄新 马占鸿 陈洪俊 李明福 |
| 作者单位： | 1. 中国检验检疫科学研究院,动植物检疫实验所,北京,100029;中国农业大学,植物检疫系,北京,100094 2. 中国检验检疫科学研究院,动植物检疫实验所,北京,100029 3. 中国农业大学,植物检疫系,北京,100094 |
| 基金项目： | 国家质检总局基金，国家"十一五"科技支撑计划 |
| 摘 要： | 目的:建立特异、灵敏、快速的TaqMam实时荧光定量PCR方法,用于烟草环斑病毒(TRSV)的定量检测。方法:用纳米磁珠法提取病毒RNA,构建包含烟草环斑病毒全CP序列的质粒标准品。根据CP保守序列设计特异性的引物和TaqMam荧光探针,构建标准曲线,建立TRSV的实时荧光绝对定量PCR方法,并对该方法的特异性、灵敏度和重复性进行评估。结果:建立的方法特异性好,与南芥菜花叶病毒、马铃薯X病毒和马铃薯Y病毒均无交叉反应;至少能检测到767个病毒拷贝,灵敏度比普通PCR高100倍;同一样品试验内及试验间重复性实验的变异系数均小于3%,重复性好;检测结果准确可靠,构建的标准曲线有较好的线性关系(R2=0.997)。结论:建立的TRSV TaqMan实时荧光定量PCR检测方法可满足口岸高通量、快速、准确的检验检疫要求。 |
| 关 键 词： | 烟草环斑病毒 实时荧光定量PCR TaqMan探针 |
Detection of Tobacco Ringspot Virus by Real-Time RT-PCR Assay |
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| LIU Mei,HUANG Xin,MA Zhan-Hon,CHEN Hong-Jun,LI Ming-Fu.Detection of Tobacco Ringspot Virus by Real-Time RT-PCR Assay[J].Letters in Biotechnology,2010,21(1):73-76. | |
| Authors: | LIU Mei HUANG Xin MA Zhan-Hon CHEN Hong-Jun LI Ming-Fu |
| Institution: | LIU Mei1,2,HUANG Xin1,MA Zhan-Hong2,CHEN Hong-Jun1,LI Ming-Fu1 1.Animal , Plant Quarantine Institute,Chinese Academy of Inspection , Quarantine,Beijing 100029,2.Department of Plant Quarantine,China Agricultural University,Beijing 100094,China |
| Abstract: | Objective:To establish a TaqMan-based real-time PCR assay for detection and quantification of Tobacco ringspot virus(TRSV).Methods:The virus RNA was extracted using viruses RNA isolation system based on magnetic nano-particles.The method was designed to use specific primers and TaqMan probe targeting conserved sequence in the coat protein gene region of TRSV down-loaded from GenBank.The specificity,sensitivity and reproducibility of the method were estimated and a standard curve was prepared.Results:The spe... |
| Keywords: | Tobacco ringspot virus real-time PCR TaqMan probe |
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