菜豆荚斑驳病毒RT-PCR扩增产物胶体金层析检测方法的建立  

Detection of Bean Pod Mottle Virus RT-PCR Amplicon by Dipstick Assay

作　　者：曹成[1] 魏梅生[2] 吴兴泉[1] 张永江[2] 李桂芬[2] 

机构地区：[1]河南工业大学生物工程学院,河南郑州450001 [2]中国检验检疫科学研究院动植物检疫研究所,北京100029

出　　处：《大豆科学》2010年第6期1024-1027,共4页Soybean Science

基　　金：国家质量监督检验检疫总局资助项目(2009IK322)

摘　　要：菜豆荚斑驳病毒是我国进境植物检疫性有害生物,为了能在现场快速检测出该病毒,将PCR核酸扩增的高灵敏度和胶体金层析方法快速简便、直观的优点结合起来,建立了PCR扩增产物的胶体金层析检测方法。该方法是用生物素标记上一个引物,用荧光素（或地高辛）标记上另一个引物,通过PCR扩增产生双标记的扩增产物。将兔抗生物素多克隆抗体与20-30 nm大小的胶体金颗粒结合上并固定在释放垫上,同时在硝酸纤维素层析膜上点上抗荧光素（或地高辛）的单克隆抗体作为T检测点,点上羊抗兔多克隆抗体作为C质控点。这种RT-PCR扩增产物可在T点上被检测到,同时C点也要显色。用所建立方法在15 min内可检测出菜豆荚斑驳病毒的RT-PCR扩增产物,免去了溴化乙锭染色和电泳的过程。Bean pod mottle virus（BPMV） is a quarantine pest for China.In order to detect the virus on site,we have developed a rapid method for detection of RT-PCR amplicon of the virus.The method is performed by labeling one prime with biotin the other primer with fluorescein（or digoxigenin）.The dual-labeled amplicon can be generated from routine polymerase chain reaction.Rabbit polyclonal antibody against biotin is conjugated with 20~30 nm colloidal gold particles fixed in a conjugate pad.Monoclonal antibody against fluorescein（or digoxigenin） is spotted on nitrocellulose membrane as T dot.Goat anti-rabbit polyclonal antibody is spotted on nitrocellulose membrane as C dot.The RT-PCR amplicon is detected on the test dot（T dot）,while the C dot serves as a control.Using this method we have detected the RT-PCR amplicon of bean pod mottle virus within 15 minutes without the staining of ethidium bromide and the running of agarose gel.

关 键 词：胶体金 试纸条 菜豆荚斑驳病毒 RT-PCR 核酸检测 

分 类 号：S436.43[农业科学—农业昆虫与害虫防治]