齿兰环斑病毒RT-qPCR检测方法的建立  

Establishment of RT-qPCR Method for Detecting Odontoglossum ringspot Virus(ORSV)

作　　者：徐匆 黄皓 黄晓彦 陈彦 李昀哲 郑芝波 XU Cong;HUANG Hao;HUANG Xiaoyan;CHEN Yan;LI Yunzhe;ZHENG Zhibo(Dongguan Center for Agricultural Science Research,Dongguan,Guangdong 523086,China)

机构地区：[1]东莞市农业科学研究中心,广东东莞523086

出　　处：《贵州农业科学》2023年第4期86-92,共7页Guizhou Agricultural Sciences

基　　金：东莞市社会科技发展(重点)项目(2020507101164)。

摘　　要：【目的】针对齿兰环斑病毒(Odontoglossum ringspot virus,ORSV)建立快速、有效检测方法,对于保障兰花规模化生产有重要意义。【方法】以齿兰环斑病毒为材料,根据NCBI上已登录的齿兰环斑病毒外壳蛋白(coat protein,cp)基因序列分析其序列保守区,使用Primer Premier 5.0设计反转录荧光定量PCR引物及TaqMan荧光探针,建立检测齿兰环斑病毒的一步法RT-qPCR。构建齿兰环斑病毒cp基因克隆质粒,并以该质粒作为标准品进行荧光定量PCR标准曲线测定;以齿兰环斑病毒、建兰花叶病毒(Cymbidium mosaic virus,CymMV)、黄瓜花叶病毒(Cucumber mosaic virus,CMV)的病毒RNA为模板进行一步法RT-qPCR特异性检测;以10倍倍比稀释的cp基因质粒为模板进行灵敏性检测;用该法对大田18个兰花样品进行齿兰环斑病毒感染情况监测。【结果】齿兰环斑病毒一步法RT-qPCR检测法仅检出齿兰环斑病毒阳性模板,其余模板检测结果为阴性,说明检测方法具有特异性;该方法能够检测到的齿兰环斑病毒cp基因最低拷贝数为130;大田兰花样品感染齿兰环斑病毒的检出率为100%。【结论】一步法RT-qPCR是一种特异性强、灵敏度高,且适合监测实际生产过程中兰花感染齿兰环斑病毒情况的检测方法。【Objective】Establishing a rapid and effective detection method for Odontoglossum ringspot virus(ORSV)means a great significance to ensure the large-scale production of orchid.【Method】ORSV is used as experimental materials.Based on the conservative region of the ORSV coat protein(cp)gene sequence registered on NCBI,reverse transcription quantitative PCR primers and TaqMan fluorescent probe are designed by Primer Premier 5.0,and one-step RT-qPCR detection for ORSV is established.The cp gene plasmid is used as the standard sample to determine the standard curve,and the specific detection of one-step RT-qPCR is carried out with the viral RNA of ORSV,Cymbidium mosaic virus(CymMV)and cucumber mosaic virus(CMV)as templates;the sensitivity is tested with 10 times diluted cp gene plasmid as templates;18 orchid samples are detected to monitor the infectious status of ORSV by the established method.【Result】The one-step RT-qPCR method can only detect ORSV positive template,and the detection results of other templates are negative,indicating that the detection method is specific.The detection limit of ORSV cp gene is 130 copies;The detection rate of orchid infected with ORSV in field is 100%.【Conclusion】The one-step RT-qPCR method is featured with specificity and sensitivity,which is suitable for monitoring the orchids infected with ORSV in the actual production process.

关 键 词：兰花 齿兰环斑病毒(ORSV) 反转录-荧光定量PCR(RT-qPCR) 外壳蛋白(cp)基因 

分 类 号：S682.31[农业科学—观赏园艺]