基于镧系高敏荧光的生物毒素现场快速检测方法的研究  

On-site rapid detection method of biotoxins based on high-sensitivity lanthanide fluorescence

作　　者：刘军波 金志颖 白雪欣 万伟[1] 杨浩 李岩伟 吴俊清 章健 高姗 王景林 LIU Jun-bo;JIN Zhi-ying;BAI Xue-xin;WAN Wei;YANG Hao;LI Yan-wei;WU Jun-qing;ZHANG Jian;GAO Shan;WANG Jing-lin(State Key Laboratory of Pathogen and Biosecurity,Institute of Microbiology and Epidemiology,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100071,China;College of Life Sciences,Hebei Normal University,Hebei Key Laboratory of Animal Physiology,Biochemistry and Molecular Biology,Shijiazhuang 050024,China;Chengdu Wellray Bio-technology Co.,Ltd,Chengdu 611730,China)

机构地区：[1]军事科学院军事医学研究院微生物流行病研究所,病原微生物安全国家重点实验室,北京100071 [2]河北师范大学生命科学学院,河北省动物生理生化与分子生物学重点实验室,石家庄050024 [3]成都微瑞生物科技有限公司,成都611730

出　　处：《军事医学》2021年第7期537-542,547,共7页Military Medical Sciences

基　　金：国家十三五生物安全重点研发专项(2018YFC1200501);国家重点实验室自主课题(SKLPBS1812)。

摘　　要：目的利用时间分辨荧光技术建立一种可快速、准确地对蓖麻毒素、A型肉毒毒素及产气荚膜梭菌ε毒素进行定量检测的方法。方法首先合成了一种可见光激发的镧系荧光微球,通过比较不同粒径的铕离子荧光微球,筛选出最佳粒径的微球偶联标记效价最高的捕获抗体,分别与相应检测抗体平行组合,确定最优的配伍组合条件建立检测方法。其次,对该法的敏感性、精密性、定量能力和特异性进行评价。结果所建立的时间分辨荧光免疫层析法(TRFIC)可在20 min内完成对3种生物毒素的定性/定量检测。3种毒素的最低检出限均可达0.5 ng/ml,定量范围为0.5~50 ng/ml,3种毒素间及与黄曲霉毒素B_(1)、T-2毒素均无交叉反应,批内重复性和批间重复性变异系数均小于15%。结论该研究建立了一种基于可见光激发的TRFIC,在生物毒素的检测中获得了较高的最低检测限,与配套的小型便携式检测仪器一起使用,具有检测快速、可远程传输结果的优势,未来在生物反恐、生物武器核查等领域具有较高的应用价值和应用前景。Objective To establish a rapid and accurate method for quantitative detection of ricin toxin,botulism toxin type A and Clostridium perfringens ε toxin by using time-resolved fluorescent technology.Methods Firstly,a type of lanthanide fluorescent microsphere excited by visible light was synthesized.By comparing the europium ion fluorescent microspheres with different particle diameters,the capture antibody with the highest titer was screened out.The optical combination condition was determined and the detection method was established.Secondly,the sensitivity,precision,quantitative ability and specificity of the method were evaluated.Results The time-resolved fluorescence immunochromatographic method established in this study could complete the quantitative detection of the three biotoxins within 20 minutes.The detection limit of the three toxins was 0.5 ng/ml,and the quantitative range was 0.5-50 ng/ml.There was no cross-reaction between the three toxins and with aflatoxin B_(1) and T-2 toxins.The coefficient of variation of intra-and inter-assay repeatability was less than 15%.Conclusion In this study,a time-resolved fluorescence immunochromatographic detection method excited by visible light is established,and a high minimum detection limit is obtained in the detection of biological toxins.It can be used in conjunction with the supporting small portable detection instrument,and has the advantages of rapid detection and remote transmission of results.In the future,it can be widely used in the fields of biological counterterrorism and biological weapon verification.

关 键 词：蓖麻毒素 A型肉毒毒素 产气荚膜梭菌ε毒素 镧系荧光 免疫层析 生物安全 

分 类 号：Q93-332[生物学—微生物学]