RT-PCR检测李坏死环斑病毒的研究  

Detection of Prunus necrotic ringspot virus(PNRSV)by PT PCR.

作　　者：孔宝华[1] 陈海如[1] 常胜军[2] 朱水芳[3] 黄文胜[3] 刘进元 

机构地区：[1]云南省植物病理重点实验室,昆明650201 [2]清华大学生命科学与工程研究院 [3]农业部植物检疫实验所

出　　处：《植物检疫》2000年第5期257-260,共4页Plant Quarantine

基　　金：云南省院省校合作基金项目

摘　　要：为了从植物组织中快速检测李坏死环斑病毒 (PNRSV) ,根据该病毒RNA 3序列设计引物 ,对感病和健康组织总RNA进行RT PCR ,结果从感病组织中扩增出了大约 45 0bp的目的片段 ,而健康组织中无此扩增带。将此PCR产物连接到 pGEM T easy载体 ,转化大肠杆菌DH5 5α菌株 ,得到了含有目的片段的重组子 ,并采用双脱氧终止法进行序列分析 ,结果与美国报道的李坏死环斑病毒RNA3序列对应部分核苷酸基本一致 (其同源性达 93.6 %) ,这表明应用RT PCR来检测李坏死环斑病毒是可行的 ;PCR产物克隆作为RT PCR反应的阳性对照解决了检疫对象不能扩散的问题 ,从而建立了RTFor rapid detection PNRSV from plant tissue,a pairs of primers were designed,corresponding the sequence of PNRSV RNA 3.The RT PCR was gotten on with total RNA of the infected tissue and health tissue.The target fragment about 450bp was amplified from the infected sample,but not from health sample.The PCR product was cloned into PGEM T easy Vector,and transformed the DH5α.The recombinant plasmid was gotten and its sequence was analyzed with Sanger method.The result is that two sequences have shown basic concert(93.6%homology),comparing the PCR product sequence with the sequence of PNRSV RNA 3.It's proved that the detection of PNRSV by RT PCR is feasible,It has resolved the problem which quarantine object can not be diffused that the PCR clone regard as PCR positive control.So the rapid detection method by RT PCR is built.

关 键 词：李坏死环斑病毒 RT-PCR 品岸植物检疫 

分 类 号：Q939.4[生物学—微生物学]