双重胶体金层析法快速检测番茄环斑病毒和烟草环斑病毒RT-PCR扩增子  

Duplex Detection of Tomato ringspot virus and Tobacco ringspot virus RT-PCR Amplicons by Dipstick Assay

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,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：《中国农学通报》2011年第22期197-201,共5页Chinese Agricultural Science Bulletin

基　　金：国家质量监督检验检疫总局课题"检疫性植物病毒核酸层析检测技术研究"(2009IK322)

摘　　要：番茄环斑病毒和烟草环斑病毒是中国进境检疫性有害生物,此研究建立了单个和双重胶体金层析快速检测方法,在15min即可获得对双标记PCR产物的检测结果。单个胶体金层析是在一张层析膜上点上荧光素(或地高辛)的单克隆抗体作为T检测点,经生物素-荧光素(或生物素-地高辛)双标记的RT-PCR扩增产物可在T点上被检测到。双重胶体金层析是在一张层析膜上分别点上荧光素和地高辛的单克隆抗体作为T1和T2检测点,经生物素-荧光素和生物素-地高辛双标记的两种病毒的RT-PCR扩增产物可分别在T1和T2点上被检测到。Both Tomato ringspot virus（ToRSV）and Tobacco ringspot virus（TRSV） are quarantine pests for China.We have developed dipstick assay for detection of RT-PCR amplicons of the two viruses within 15 minutes.The single dipstick assay is performed by labeling primers of the virus with biotin and fluorescein（or digoxigenin）.Monoclonal antibody against fluorescein（or digoxigenin） is spotted on nitrocellulose membrane as T dot.The dual-labeled（biotin-fluorescein or biotin-digoxigenin） RT-PCR amplicons can be detected on the test dot（T dot）.The duplex dipstick assay is performed by labeling primers of ToRSV with biotin and fluorescein（or digoxigenin） and primers of TRSV with biotin and digoxigenin（or fluorescein）.Monoclonal antibody against fluorescein and digoxigenin is spotted on nitrocellulose membrane respectively as T1 and T2 dot.The dual-labeled（biotin-fluorescein and biotin-digoxigenin） RT-PCR amplicons of the two viruses can be detected on the T1 and T2 dot simultaneously.

关 键 词：番茄环斑病毒 烟草环斑病毒 RT-PCR 胶体金 双重检测 

分 类 号：S41-30[农业科学—植物保护]