草莓斑驳病毒的分子杂交检测

Detection of Strawberry mottle virus by molecular hybridization

为探索分子杂交技术在草莓斑驳病毒(Strawberry mottle virus,SMoV)检测中的运用,利用CTAB法从草莓叶片中提取总核酸,并以此为模板,利用特异引物扩增SMoV非编码区序列,经克隆、测序获得SMoV非编码区序列。系统进化分析结果显示:本研究中的分离物与GenBank中已报道的分离物的亲缘关系较远,独立形成1个小的进化支。以带有病毒特异片段的质粒为模板,运用PCR技术制备了地高辛标记的cDNA探针。以草莓感染SMoV叶片为试材,利用地高辛标记探针可有效检测草莓叶片中的SMoV,草莓总核酸样品稀释10倍后仍可有效检测。

To validate the technique of molecular hybridization on the detection of Strawberry mottle virus （SMoV）, total nucleic acid, which extracted from strawberry leaves using CTAB approach, was adopted as a template to be amplified with specific primers in non-coding region of SMoV. And then, sequences of SMoV were obtained after cloning and sequencing phases. The results of phylogenetic analysis indicated that there was a far relative between the isolate in this research and the reported isolates in GenBank, the isolate in the study formed a small unattached branch in phylogenetic tree. Using plasmid DNA with viral specific fragment as a template, a eDNA probe labeled with digoxigenin was prepared by PCR. SMoV could be efficiently detected by the cDNA probe in the test material of strawberry leaves infected by SMoV, as well as in the extreme case with 10 folds dilution solution of total nucleic acid. To our knowledge, this is the first report on the detection of SMoV by cDNA probe.