半巢式RT-PCR检测进口大豆中菜豆荚斑驳病毒的研究

 菜豆荚斑驳病毒(Bean pod mottle virus,BPMV)在美国东部和南部的大豆产区广泛分布。该病毒能够造成3%~52%的产量损失并使大豆品质降低,目前我国未见其分布。针对进口大豆种子的植物病毒检测,本文建立了半巢式RT-PCR技术检测BPMV的方法。该方法采用Trizol快速提取大豆种子病毒总RNA,并根据BPMV的外壳蛋白编码基因设计特异性引物,经第一轮RT-PCR和第二轮半巢式PCR扩增,分别得到275 bp和196 bp大小的特异性基因片段。半巢式RT-PCR扩增产物的测序表明,该产物序列与BPMV的外壳蛋白编码基因存在91%~95%的高度同源性。检测灵敏度比较研究显示,DAS-ELISA与RT-PCR的检测灵敏度相近,半巢式RT-PCR的检测灵敏度比这2种方法高出103倍以上。

Detection of Bean pod mottle virus by seminested RT-PCR in imported soybean

Bean pod mottle virus(BPMV) is widespread in the major soybean-growing areas in the east and south of the United States,which caused 3%-52% yield reductions and poor seed quality.Up to date,BPMV is not distributed in our country.For the purpose of BPMV detection in imported soybean seeds,we adoppted Trizol for total RNA extraction and developed a semi-nested RT-PCR method with three specific primers,designed on the basis of coding genes of BPMV coat protein.Aplicons of RT-PCR and semi-nested PCR were 275 bp and 196 bp,respectively.Sequencing of the semi-nested RT-PCR product showed its identities with the coding genes of BPMV coat protein were 91%-95%.Further study indicated that detection sensitivities of DAS-ELISA and RT-PCR methods were about the same,but that of semi-nested RT-PCR was 10~3 times higher at least.