南芥菜花叶病毒RT-PCR扩增产物胶体金层析和酶联检测方法的建立  

Detection of Arabis mosaic virus RT -PCR amplicons by dipstick assay and ELISA

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

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

出　　处：《植物检疫》2011年第5期33-36,共4页Plant Quarantine

基　　金：国家质量监督检验检疫总局课题(2009IK322)

摘　　要：南芥菜花叶病毒是我国进境植物检疫性有害生物,为建立快速灵敏的检疫检测方法,本研究用生物素标记一条引物,用荧光素(或地高辛)标记另一条引物,经RT-PCR扩增产生双标记的扩增产物,建立了PCR扩增产物的胶体金层析检测和ELISA检测方法。胶体金层析检测方法在15min即可获得检测结果,ELISA方法需20h,后者检测的灵敏度可比前者分别提高100倍和5000倍。所建立的2种方法都免去了普通PCR检测的溴化乙锭染色和电泳的过程。Arabis mosaic virus （ArMV） is a quarantine pest for China. We have developed a rapid method for detection of RT - PCR amplicons of the virus by dipstick assay. The method is performed by labeling one primer with biotin the other primer with fluorescein （ or digoxigenin）. The dual - labeled amplicons can be generated from routine polymcrase chain reaction. Rabbit polyclonal antibody against biotin is conjugated with 20 - 30 nm colloidal gold particles. 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 amplicons are detected on the test dot（ T dot）, while the C dot serves as a control. Using this method we have detected the RT -PCR amplicons of ArMV within 15 minutes. The dual -labeled amplicons can also be detected by ELISA. Both the dipstick and ELISA can detect the dual - labeled amplicons without the staining of ethidium bromide and the running of agarose gel. ELISA is more sensitive than dipstick assay.

关 键 词：南芥菜花叶病毒 胶体金 试纸条 ELISA 核酸检测 

分 类 号：S41-30[农业科学—植物保护]