进口大豆上菜豆荚斑驳病毒的免疫捕获巢式RT-PCR检测  

Detection of Bean pod mottle virus by immuno-capture nested RT-PCR from the imported soybean

作　　者：于翠[1] 杨翠云[1] 宋绍祎[1] 洪健[2] 

机构地区：[1]上海出入境检验检疫局,200135 [2]浙江大学分析测试中心

出　　处：《植物检疫》2006年第4期201-204,共4页Plant Quarantine

摘　　要：利用DASELISA对进口大豆上BPMV进行检测发现,从美国进口的部分大豆样品对BPMV的多抗血清呈阳性反应;利用免疫吸附电镜观察发现,ELISA检测阳性的大豆种皮病汁液中存在直径约30nm的球状病毒粒子。根据BPMV外壳蛋白(CP)基因的保守序列设计了2对嵌合引物,建立了BPMV的高灵敏的免疫捕获巢式RTPCR(ICnestedRTPCR)检测方法。该方法经免疫捕获、反转录和2轮PCR扩增,能从带毒大豆种子中扩增到预期大小的DNA条带。序列测定与分析表明此条带的序列为BPMV部分CP基因,在系统关系树上与BPMV的其它分离物形成一簇亲缘关系很近。实验表明从进境大豆上检测到了BPMV。For detection Bean pod mottle virus （BPMV）from the imported soybean（ Glycine max）seeds, double anti- body sandwich-ELISA was performed and the results showed that a part of soybean samples imported from USA produced postive reations with polyclonal antibody against BPMV. Isometric viral particles of about 30 nm in diameter were observed from infected soybean seed coats by immuno-sorbent electron microscopy. Based on the conserved sequences of BPMV coat potein （CP）gene, two primer pairs were designed and an immuno-capture nested RT-PCR. This method consists of three major steps ：immuno-caputer, reverse transcription and two times of PCR ampilification and produced a specific band with a expected size from the infected soybean seeds. Sequence determination and analysis show that the determined sequence is the partial CP gene of BPMV, and it forms one branch on the phylogenetic tree and has the closest relationship with other isolates of BPMV, indicating that Bean pod mottle viurs was detected from the imported soybean seed.

关 键 词：菜豆荚斑驳病毒 大豆 ELISA 免疫电镜 免疫捕获巢式 RT-PCR 

分 类 号：S41-3[农业科学—植物保护]