四种广普性植物病毒高效mPCR检测方法的建立  

A simple and Reliable Assay for Detecting Four Broad-spectrum Plant Viruses Using Multiplex Polymerase Chain Reaction Method

作　　者：曾静[1] 张飞云[1] 李晶晶[1] 于银霞[1] 王法军[1] 

机构地区：[1]首都师范大学,北京100037

出　　处：《生物技术通报》2007年第6期108-112,共5页Biotechnology Bulletin

基　　金：国家自然科学基金面上项目(30571010);教育部重点项目(204006);北京市教委科技发展计划项目(KM200310028102);教育部留学归国人员启动经费

摘　　要：本研究建立了能同时检测出烟草花叶病毒(TMV)、黄瓜花叶病毒(CMV)、马铃薯X病毒(PVX)和马铃薯Y病毒(PVY)的多重RT-PCR体系。TMV、CMV、PVX、PVY是四种广普性植物病毒,寄主范围广泛,并且常常发生复合侵染。本研究以上述四种病毒的CP基因部分序列设计引物,以反转录的cDNA为模板,建立多重RT-PCR反应体系,分别扩增出211～417bp的不同长度的基因片断,并通过序列测定来确认扩增序列的特异性。将反转录合成的cDNA进行浓度稀释,来对多重RT-PCR与单重RT-PCR的灵敏度进行比较,结果证明,多重RT-PCR体系能够同时快速检测这四种病毒,并且有很高的灵敏度。A multiplex polymerase chain reaction（mPCR）assay was developed to detect four RNA broad-spectrum plant viruses：Tobacco mosaic virus（ TMV ） , Cucumber mosaic virus（ CMV ） ,Potato virus X（PVX）and Potato virus Y（PVY）. The hosts of the four viruses are very broad,and often complex infected. In the present research,the primers were designed based on the respective virus isolate sequence data available from the GeneBank and were used for reliable detection of the four viruses by simplex-and multiplex RT-PCR. Four different length fragments（211-417 bp）specific to the viruses were simultaneously amplified using mPCR and were identified on the basis of their molecular sizes. The mPCR results were confirmed with sequencing analysis. In addition,the results of the mPCR were compared with simplex PCR by diluting the viruses＇ cDNA,which proved to be a useful rapid method for detecting the four broadspectrum plant viruses.

关 键 词：多重RT-PCR 烟草花叶病毒 黄瓜花叶病毒 马铃薯X病毒 马铃薯Y病毒分子检测 

分 类 号：S432.41[农业科学—植物病理学]