烟草环斑病毒和番茄环斑病毒的半巢式RT-PCR检测  

Detection of Tobacco ringspot virus and Tomato ringspot virus by semi-nested RT-PCR

作　　者：闻伟刚[1] 崔俊霞[1] 盛蕾[1] 

机构地区：[1]宁波出入境检验检疫局,浙江宁波315012

出　　处：《植物保护学报》2007年第1期61-66,共6页Journal of Plant Protection

基　　金：宁波出入境检验检疫局科技项目(甬K04-2005)

摘　　要：烟草环斑病毒（Tobacco ringspot virus，TRSV）和番茄环斑病毒（Tomato ringspot virus，ToRSV）是我国禁止入境的检疫性有害生物。采用半巢式RT．PCR方法对这两种病毒进行了检测，用Trizol快速提取病毒总RNA，并根据TRSV和ToRSV的外壳蛋白基因设计特异性引物，经过第一轮RT-PCR和第二轮半巢式PCR扩增，TRSV样本分别得到359bp和206bp特异性片段，TORsV样本分别得到340bp和219bp特异性片段。半巢式RT．PCR扩增产物的测序表明，TRSV产物序列与GenBank中登录的外壳蛋白基因存在88％～97％的同源性，TORSV产物序列与GenBank中登录的外壳蛋白基因存在96％～100％的同源性。研究显示，DAS-ELISA与RT-PCR的检测灵敏度相近，而半巢式RT-PCR的检测灵敏度比这两种方法高出10^3倍以上。Tobacco ringspot virus （TRSV） and Tomato ringspot virus （ToRSV） are quarantine pests. For the detection of these two viruses, we adopted Trizol for total RNA extraction and developed a semi-nested RT-PCR method with its specific primers, which designed on the basis of the coding genes of TRSV and ToRSV coat protein, respectively. Products of TRSV by RT-PCR and semi-nested PCR were 359 bp and 206 bp, that of ToRSV were 340 bp and 219 bp. Sequencing of the semi-nested RT-PCR products of TRSV and ToRSV indicated that their identities with the TRSV and ToRSV coat protein genes were 88% -97% and 96% -100%, respectively. Further study showed that the detection limits of DAS-ELISA and RT-PCR methods were about the same, but 10^3 times lower than that of the semi-nested RT-PCR at least.

关 键 词：烟草环斑病毒 番茄环斑病毒 半巢式RT-PCR 检测灵敏度 

分 类 号：S432.4[农业科学—植物病理学]