环介导等温扩增技术与横向流动试纸条法快速检测鳗利斯顿氏菌的研究  

Rapid Detection of a Bactereium Listonella anguillarum by Loop-mediated Isothermal Amplification Combined with a Lateral Flow Dipstick Method

作　　者：董培培[1] 李长红[1] 丁文超[1] 陈炯[1] 史雨红[1] 黎昊雁 李明云[1] 

机构地区：[1]宁波大学应用海洋生物技术教育部重点实验室,浙江宁波315211 [2]浙江省出入境检验检疫局,浙江杭州310012

出　　处：《水产科学》2011年第2期63-68,共6页Fisheries Science

基　　金：浙江省科技厅重点项目(2008C22049;2008R40G2070059);长江学者和创新团队发展计划项目(IRT0734)

摘　　要：采用环介导等温扩增技术(Loop-mediated isothermal amplification,LAMP)、横向流动试纸条技术(Lateral flow dipstick,LFD),建立一种鳗利斯顿氏菌快速检测方法。针对鳗利斯顿氏菌金属蛋白酶基因设计一套特异性引物及异硫氰酸荧光素(Fluorescein isothiocyanate,FITC)标记的探针,结合生物素标记的环介导等温扩增技术扩增反应和横向流动试纸条技术对鳗利斯顿氏菌进行检测;比较LAMP-AGE、LAMP-LFD和PCR-AGE的灵敏度;选取4株鳗利斯顿氏菌和6株非鳗利斯顿氏菌验证LAMP-LFD特异性。试验结果表明,应用LAMP-LFD,能在30 min内完成鳗利斯顿氏菌的检测;LAMP-LFD检测的灵敏度为7.7 cfu/ml,LAMP-AGE和PCR-AGE检测的灵敏度均为77 cfu/ml;4株鳗利斯顿氏菌均呈阳性反应,其他6株非鳗利斯顿氏菌均为阴性。应用LAMP-LFD检测鳗利斯顿氏菌特异性强、灵敏度高、并且操作安全、简便、快捷。A rapid detection of a vibro Listonella anguillarum was developed by loop-mediated isothermal amplification（LAMP）combined with a lateral flow dipstick（LFD）method.A set of six primers and a fluorescein isothiocyanate（FITC）-labeled DNA probe were designed specifically to recognize the metalloprotease empA gene in the vibro.The LAMP method for amplification of the metalloprotease empA gene in the vibro using biotin-labeled primer was combined with a chromatographic LFD for rapid and simple visual detection of the vibro-specific amplifications.The sensitivity of LAMP combined with agarose gel electrophoresis（LAMP-AGE）,LAMP-LFD and PCR combined with agarose gel electrophoresis（PCR-AGE）were compared.Four L.anguillarum strains and six non-L.anguillarum strains were selected to confirm the specifity of LAMP-LFD.Using the combined LAMP and LFD system,the total assay interval was approximately 30 min,excluding the time for DNA extraction.The detection limit of L.anguillarum by LAMP-LFD was 7.7 cfu per ml,but LAMP-AGE and PCR-AGE could detect up to 77 cfu per ml.All four L.anguillarum strains gave a positive reaction,whereas no amplification was observed in other six non-L.anguillarum strains.The detection of L.anguillarum by LAMP-LFD is a safe,quick and effective procedure with high specificity,sensitivity,and feasibility.

关 键 词：鳗利斯顿氏菌 empA基因 环介导等温扩增技术 横向流动试纸条技术 检测 

分 类 号：S917.1[农业科学—水产科学]