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.
出处
《植物检疫》
北大核心
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)
