H5、H7和H9亚型禽流感病毒液相芯片检测方法的建立  

Establishment of a liquid chip method for detection of avian influenza virus subtypes H5,H7 and H9

作　　者：张晓娜[1,2] 王慧煜[1] 梅琳[1] 韩雪清[1] 林祥梅[1] 张书霞[2] 

机构地区：[1]中国检验检疫科学研究院,北京100029 [2]南京农业大学动物医学院,江苏南京210095

出　　处：《中国兽医科学》2013年第5期494-499,共6页Chinese Veterinary Science

基　　金：国家重点基础研究发展计划(973)项目(2011CB504704)

摘　　要：为应用液相芯片技术建立同时检测H5、H7、H9亚型禽流感病毒(AIV)的方法,在GenBank中收集H5、H7、H9亚型AIV的HA基因序列,利用DNAMAN软件分析比较筛选出特异的保守片段,设计引物和探针。将多重不对称RT-PCR扩增产物与偶联荧光微球的探针进行杂交,建立多重液相芯片方法。结果显示,多重液相芯片技术对H5、H7、H9亚型AIV核酸的最低检出量分别为18.5、28.7、20.7pg/μL,检测的特异性和重复性都很好,应用该方法和禽流感检测试剂盒双盲试验同时对50份已知病毒的样品进行了检测,两者符合率为100%。该方法为禽流感病毒核酸液相芯片的进一步研究奠定了基础。To establish a liquid chip method based on bio-plex suspension array for detection of avian influenza virus（AIV） subtypes H5, H7 and H9, HA gene sequences of three AIV subtypes were retrieved from GenBank,and conserved regions were selected to design specific primers and probes using DNAMAN software. PCR products by multiplex asymmetric RT-PCR and probes which were conjugated with the cor- responding fluorescent microspheres were hybridizated to establish a multiplex liquid chip detection method. The result showed that this method was specific and sensitive and the detectable level could be 18.5 pg/μL of H5,28. 7 pg/μL of H7 and 20.7 pg/μL of H9, respectively. This method and commercial AIV detection kits were applied to detect simultaneously 50 known samples under double-blind test and the two results were the same. This method provides the foundation for further study of liquid chip for detection of AIV.

关 键 词：液相芯片 核酸杂交 禽流感病毒 快速检测 

分 类 号：S852.659.5[农业科学—基础兽医学]