双重一步法RT-PCR检测2种主要兰花病毒  

Detection of Two Main Orchid Viruses with the Method of Double One-step RT-PCR

作　　者：柳爱春[1] 赵芸[1] 刘超[1] 张乐[1] 

机构地区：[1]杭州市农业科学研究院,浙江杭州310024

出　　处：《安徽农业科学》2009年第27期12960-12961,13009,共3页Journal of Anhui Agricultural Sciences

基　　金：杭州市科技发展计划项目(20070132N09)

摘　　要：[目的]研究建兰花叶病毒(CymMV)和齿兰环斑病毒(ORSV)双重一步法RT-PCR检测方法,为病毒的早期快速诊断提供技术支持。[方法]根据2种病毒的外壳蛋白基因的保守序列分别设计两对特异性引物,并进行双重与单一RT-PCR对比试验。[结果]结果在健康蜘蛛兰被人工接种病毒后第10天和第21天用一步法RT-PCR方法分别获得CymMV和ORSV与预期相符的约764和946 bp扩增产物条带;且双重一步法RT-PCR产物经琼脂糖凝胶电泳后,出现两条与单一RT-PCR产物相对应的清晰条带,获得较理想的检出效果;扩增产物的核酸序列经BLAST分析,同源性均在85%以上,证实了检测结果的准确性。[结论]该方法具有更加灵敏、特异、快速和简便的特点,适合于两种主要兰花病毒的同时快速检测。[ Objective ] The technical support for the rapid diagnosis of orchard virus in early period was provided through the establishment of the detection method of double one-step RT-PCR for Cymbidium mosaic virus（CymMV） and Odontoglossum ringspot virus（ORSV）. [ Method ] According to the two virus coat protein gene conservative sequence, of two pairs of specific primers were designed, respectively, and the experiment in the double and single RT-PCR were compared. [ Results] The results indicated that the 764 bp and 946 bp of amplified product bands from the healthy spider orchid inoculated with virus on the 10th and 21st day were matched with those of CymMV and ORSV with the method of one-step RT-PCR, respectively; and two clear bands with method of double one-step RT-PCR after agarose gel electrophoresis was formed corresponding single RT-PCR product, which was with better detection results. The homology of the nucleic acid sequences of amplified products was more than 85% by BLAST analysis, which accuracy of test results was further confirmed. [ Conclusion] The method is more sensitive, specific, rapid and convenient, and suitable for the rapid detection of virus of two main types of orchids at the same time.

关 键 词：建兰花叶病毒 齿兰环斑病毒 双重一步法RT—PCR 

分 类 号：S188[农业科学—农业基础科学]