禽流感病毒通用型RT-LAMP快速检测方法的建立  

Development of a loop-mediated isothermal amplification method for detection of alldifferent subtype avian influenza virus

作　　者：石霖[1,2,3] 王秀荣[2] 孙阮洋 李雁冰[2] 程荣华[4] 于学武[3] 曹东[3] 薛树山[3] 赵培[3] 徐晓龙[2] 马勇[2] 郑世民[1] 

机构地区：[1]东北农业大学动物医学学院,黑龙江哈尔滨150030 [2]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/农业部动物流感重点开放实验室,黑龙江哈尔滨150001 [3]辽宁省动物疫病预防控制中心,辽宁沈阳110164 [4]吉林省畜牧兽医科学研究院,吉林长春130062

出　　处：《中国预防兽医学报》2015年第5期383-386,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：十二五农村领域国家科技课题(2012AA101303);国际合作项目(2014DFR31260);吉林省社会公益研究行业科研专项(2060302)

摘　　要：为建立一种禽流感病毒（AIV）的快速检测方法,本研究根据环介导等温扩增技术（LAMP）原理,设计了针对AIV M基因4个不同区段的特异性引物,并对反应条件和反应体系进行优化,建立了AIV的RT-LAMP方法。结果表明,该方法对AIV H1~H16亚型的检测结果均为阳性,而对其他禽类病毒的扩增均为阴性,具有良好的特异性。对AIV的最低检测限为13.43 EID50/m L,灵敏度为普通一步RT-PCR的10倍;扩增反应可以在恒温水浴内60 min内完成;在反应体系中添加荧光染料后,肉眼即可判定检测结果。该检测方法与RT-PCR方法和荧光定量RT-PCR方法对临床样品检测的符合率均为81.8%。该方法能够简便、快速、灵敏、特异地检测AIV。In order to establish a rapid detection assay for all different subtype avian influenza virus （AIV）, the reverse transcription loop-mediated isothermal amplification （RT-LAMP） was developed with a set of primers designed according to 4 regions of the virus M gene. The results showed that the RT-LAMP was specific to amplification from H1 to H16 different subtype AIVs, but no cross-reaction to other avain viruses. The detection limit of the RT-LAMP assay was 13.43 EIDJmL for AIV, which was 10-fold sensitive than that of one-step RT-PCR. The amplification was completed within 60 rain in a water both, and the amplified results was able to be visualized by adding fluorescein in the reactions. The RT-LAMP detection method was evaluated by comparison with RT-PCR and real-time RT-PCR for detection of clinical samples, the coincidence of positive rate was 81.8%. These results demonstrated that the RT-LAMP method is a sensitive and rapid method for application in field diagnosis AIV.

关 键 词：禽流感 RT-LAMP 诊断方法 

分 类 号：S852.65[农业科学—基础兽医学]