H9亚型禽流感病毒RT-PCR检测方法的建立  

Development of one step RT-PCR assay for detection of avian influenza virus subtype H9

作　　者：包红梅[1] 王秀荣[1] 陶启蒙[1] 蔡东东[1] 王馥梅[1] 赵玉辉[1] 陈化兰[1] 

机构地区：[1]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

出　　处：《中国兽医科学》2010年第4期384-389,共6页Chinese Veterinary Science

基　　金：防禽流感高效生物制剂试生产开发项目(2007DFR30360)

摘　　要：通过分析流感数据库中123个HA序列,根据HA保守区序列设计并合成了1对引物,建立了一步法RT-PCR检测方法,预期扩增片段大小为732 bp。通过对H9亚型禽流感病毒（AIV）不同稀释度的尿囊液和棉拭子浸出液进行检测,证实病毒尿囊液的最低检出量为1×10^4.7EID50/mL;阳性棉拭子的最低检出量为1×10^2.5EID50/mL。用该方法检测H1-H15亚型AIV和鸡新城疫病毒等其他14种禽病病原,结果仅有H9亚型AIV出现特异性目的条带,而其他均未出现目的条带。脏器及咽喉、泄殖腔棉拭子样品在1∶10稀释度下,病毒分离和RT-PCR方法可以达到相同的阳性检出率。表明建立的AIV H9亚型RT-PCR方法具有较好的特异性和敏感性。According to 123 hemagglutinin（HA） gene sequences of avian influenza virus（AIV） subtype H9,a pair of specific oligonucleotide primers was designed.One step RT-PCR assay was developed with the primers for the detection of AIV subtype H9.The amplification by the one step RT-PCR assay was 732bp in size.The detection limit of allantoic fluid by the one step RT-PCR reached 1×10^4.7 EID50/mL and the detection limit of swab samples reached 1×10^2.5EID50/mL.The tissue and swab samples infected with AIV subtype H9 were simultaneously tested by the one step RT-PCR and the virus isolation method.The positive rate of the infectious samples,which were diluted at 1∶10,tested by the one step RT-PCR was in accordance with that by the conventional virus isolation method.AIV subtypes H1 to H15,Newcastle disease virus and other 14 avian pathogens were tested by the one step RT-PCR.Results showed the developed assay was highly specific and sensitive for the detection of AIV subtype H9.

关 键 词：禽流感病毒 一步法RT-PCR H9亚型 

分 类 号：S852.659.5[农业科学—基础兽医学]