H7亚型禽流感病毒一步法RT-PCR检测方法的建立  

Development of one step RT-PCR technique for detection of H7 subtype avian influenza

作　　者：包红梅[1] 王秀荣[1] 陶启蒙[1] 蔡东东[1] 王馥梅[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侬业部动物流感重点开放实验室,哈尔滨150001

出　　处：《生物工程学报》2009年第11期1658-1663,共6页Chinese Journal of Biotechnology

基　　金：防禽流感高效生物制剂试生产开发项目(No.2007DFR30360)资助~~

摘　　要：通过分析流感数据库45个H7亚型禽流感病毒的HA序列,在保守区内设计并合成引物,建立了一步法RT-PCR检测方法,扩增片段大小为501bp。通过对H7亚型禽流感病毒尿囊液和棉拭子浸出液不同滴度检测,证实病毒尿囊液最低检出量为105.5EID50/mL;阳性棉拭子最低检出量为103EID50/mL。用该方法检测H1～H15亚型禽流感病毒和鸡新城疫病毒等其他14种禽病病原进行检测,仅有H7亚型AIV有特异性目的条带,与其他均无交叉反应。从脏器及咽喉、泄殖腔棉拭子样品的病毒分离和RT-PCR方法比较,表明在10-1的样品浓度下,两者可以达到相同的检出量。表明该一步法RT-PCR方法具有特异性强、敏感性高和准确率高的特点。According to 45 hemagglutinin（HA） gene sequences of H7 subtype of avian influenza virus（AIV）,a pair of specific oligonucleotide primers was designed.We developed one step RT-PCR for detecting AIV subtype H7.Sensitivity to detection of allantoic fluid by one step RT-PCR reached 105.5 EID50/mL and detection of swab samples reached 103 EID50/mL.We simultaneity detected the tissue and swab samples infected with H7 subtypes AIV by one step RT-PCR and virus isolation method.The results showed that the sensitivity of the assay gave an excellent correlation with the conventional virus isolation method.H1-H15 subtypes of avian influenza and other avian diseases were detected by the one step RT-PCR.The results showed the assays were high specific,without cross-reaction with other subtypes or other avian diseases.Development of one step RT-PCR will provide effective technical support for the rapid diagnosis and surveillance of molecular epidemiology of AIV subtype H7.

关 键 词：禽流感病毒 反转录聚合酶链反应 H7N1 亚型鉴别 

分 类 号：S852.65[农业科学—基础兽医学]