荧光定量RT-PCR检测H5亚型禽流感病毒方法的建立与验证  

Development and Validation of Real-time RT-PCR Assay for Detection of Avian Influenza Virus H5 Subtype

作　　者：曹军平[1,2] 陆桂平[1] 徐向明[1,2] 顾敏[2] 徐全刚[2] 刘武杰[2] 彭大新[2] 刘秀梵[2] 

机构地区：[1]江苏畜牧兽医职业技术学院,江苏泰州225300 [2]扬州大学兽医学院,江苏扬州225009

出　　处：《中国畜牧兽医》2011年第11期169-174,共6页China Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金重点项目(30630048);江苏省农业三项工程项目(SX(2008)092)

摘　　要：根据H5亚型禽流感病毒HA基因上的保守序列,设计合成引物,以H5亚型禽流感病毒HA基因重组质粒为标准品绘制标准曲线,建立了荧光定量逆转录聚合酶链反应检测方法。结果表明,本试验建立的标准曲线循环阈值(Ct值)与模板浓度具有良好的线性关系,相关系数为0.995,灵敏度约为8拷贝/μL,相当于8个AIV颗粒,对新城疫病毒和其他禽病病毒无交叉反应,特异性好、重复性佳,为H5亚型禽流感病毒检测提供了一种特异、敏感、快速、低成本、高通量、生物安全性好的定量检测方法。对178份临床泄殖腔棉拭子样品的检测,该方法结果与经典病毒分离方法符合率大于90.0%,在禽流感病毒临床样品快速筛检、流行病学监测等方面显示了良好的应用前景。According to the conservative region of H5 AIV HA gene,the primers were devised and synthesized.A serial 10 fold dilutions positive plasmid was prepared and used for standard.The standard curve revealed the linear relationship between Ct（cycle threshold） and template concentration with a good correlation（R2=0.995）.The RRT-PCR method for the detection of AIV was highly specific and sensitive,and it could be used for rapid quantitative detection of AIV H5 subtype.No cross-reaction was detected against other avian disease viruses.Sensitivity was 8 copies of AIV genome.The total meet rate with traditional virus isolation method was more than 90.0% in detecting 178 clinical cloacal swab samples.It showed that a real-time RT-PCR method for detection of H5 subtype avian influenza virus had been established in this study,which had properties such as high speed,specific,sensitive,cheap,high-throughput,and good biological safety as well as displaying good prospects in the rapid screening of clinical samples and epidemiological monitoring of avian influenza virus H5 subtype.

关 键 词：H5亚型禽流感病毒 荧光定量RT-PCR 建立 验证 

分 类 号：S858.31[农业科学—临床兽医学]