WSSV和IHHNV对虾病毒的胶体金免疫层析快速检测  

An easy and simple method for simultaneous detection of two viruses(WSSV and IHHNV)in penaeid shrimp by a colloidal gold immunochromatography assay

作　　者：丁东 熊云 方勇新 林标扬[2] 

机构地区：[1]杭州普望生物技术有限公司,浙江杭州310058 [2]浙江大学浙江加州国际纳米研究院,浙江杭州310018

出　　处：《水产养殖》2014年第10期1-7,共7页Journal of Aquaculture

摘　　要：为了快速、方便地检测出这2种病毒,研究将PCR核酸扩增的高灵敏度和胶体金免疫层析技术快速、简便、直观的优点结合起来,建立了同时检测2种对虾病毒（WSSV和IHHNV）的同步PCR-胶体金免疫层析检测方法.采用了生物素标记和地高辛（Digoxigenin）标记引物.PCR扩增产物利用胶体金免疫层析技术进行检测.胶体金免疫检测试剂条的检测线（T线）处的层析膜处点上地高辛的单抗,经地高辛标记的PCR扩增产物可在T线上被检测到,在10 min内即可得到检测结果.同时,在PCR过程中使用UNG（尿嘧啶-DNA糖基酶）,极大地控制了遗留污染.通过此方法检测对虾WSSV和IHHNV病毒的灵敏度均可达到10～100个拷贝,高于国家标准PCR检测法10～100倍.此检测方法让工作人员避免了接触有毒和诱导突变的试剂,免除基层检测实验室购买电泳设备和荧光检测系统的投资.White spot syndrome virus （WSSV） and infectious hypodermal and haematopoietic necrosis virus （IHHNV） are the two major pathogens that cause massive mortalities of cultured penaeid shrimps in the world. We have developed a rapid and convenient method for simultaneous detection of the two viruses by combining PCR with amplicon detection by a colloidal gold immunochromatography assay. The method starts by labeling one primer with biotin amt the other primer with digoxigenin. The dual-labeled amplicon （biotin-digoxigenin）can be detected by colloidal gold immmlochromatography assay. The antibody against digoxigenin is spotted on nitrocel- lulose membrane as T （test）line on the colloidal gold dipstick. The dual-labeled amplicon can be detected in as little as 10 minutes, a dramatic reduction of the normal two-hour turnaround time for DNA electrophoresis and ethidium bromide（ EB ）staining. We also included the use of UNG （Uracil N-glyeosylase） in the PCR reaction to minimize carryover contamination. The method has a detection sensitivity of 10-100 copies of template,which is about 10-100 times more sensitive than the current national standard detection method under the same testing conditions. Our method also averts the exposure of workers to toxic mutagens and saves the expenditure on buy- ing electrophoresis and fluorescent detection system for local site-based laboratories.

关 键 词：对虾 白斑综合征病毒 传染性皮下和造血器官坏死病毒 PCR 胶体金免疫赏析 同步检测 

分 类 号：S945.4[农业科学—水产养殖]