荧光纳米微球免疫层析技术检测新型冠状病毒研究

Detection of Novel Coronavirus by fluorescent nanomicrosphere immunochromatography

采用碳二亚胺(EDC)法将抗新冠病毒核衣壳蛋白(N蛋白)单克隆抗体与稀土铕荧光纳米微球共价偶联,合成荧光标记免疫探针,并用透射电子显微镜进行表征。开发了一种检测新型冠状病毒的荧光免疫检测试纸条,通过荧光检测仪测定检测线上的荧光强度,建立了定量检测新冠病毒的荧光免疫检测技术。本研究研制的新冠病毒荧光免疫层析试纸条,与荧光检测仪配套使用,对新冠病毒N蛋白的最低检出限达到0.01 ng/mL;对新冠病毒检测特异性强,与肺炎支原体、禽流感病毒等其它呼吸道肺炎病原物无交叉反应;对新冠灭活病毒的检测,用本研究开发的荧光免疫试纸条技术与荧光定量PCR进行比对验证,灵敏度为荧光定量PCR的1/100;与胶体金抗原检测试纸条相比较,比胶体金试纸条灵敏10倍以上。本研究开发的新冠病毒荧光检测试纸条,操作简便、快速、灵敏、成本低、体积小,适于现场快速检测。

A monoclonal antibody against Novel Coronavirus nucleocapsid protein(N protein)was covalently coupledwithrare earth europium fluorescent nanospheres by carbodiimide(EDC)method,and a fluorescent labeled immune probe was synthesized.The fluorescent labeled immuneprobes were characterized by transmission electron microscopy.A fluorescent test strip for detecting Novel Coronavirus was developed,the fluorescence intensity on the detection line was measured by the fluorescence detector,and the content of Novel Coronavirus was quantitatively measured.The fluorescence test strip developed in this study was used in conjunction with a fluorescence detector,and the detection limit of Novel Coronavirus N protein was 0.01 ng/mL.The strip detection of Novel Coronavirus had strong specificity,without any cross reaction observed in other respiratory tract pneumonia pathogenssuch as mycoplasma pneumoniae,avian influenza virus and so on.The detection results of the Novel Coronavirus inactivated virus using the fluorescence test stripswereconsistent with the fluorescence quantitative PCR with the sensitivity was 1/100 of fluorescence quantitative PCR.Compared with the colloidal gold antigen test strip,the fluorescent stripwas 10 times more sensitive than the colloidal gold test strip.The fluorescent test strip developed in this study for the detection of Novel Coronavirus is prompt,fast,sensitive,low cost,small sized and suitable for rapid on-site detection.