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期 刊 ：植物保护学报 2007年

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；

Keywords:Tobacco ringspot virus, Tomato ringspot virus, semi-nested RT-PCR, detection sensitivity,

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359bp和206bp特异性片段,ToRSV样本分别得到340bp和219bp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10³倍以上。

Abstract: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 10³ times lower than that of the semi-nested RT-PCR at least.

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