H9亚型禽流感病毒荧光定量RT-PCR检测方法的建立  

Development of a Real-time RT-PCR assay for detection of avian influenza virus H9 subtype

作　　者：陆桂平[1] 曹军平[1,2] 王涛[1] 赵明军[2] 赵国[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：《畜牧与兽医》2011年第7期1-5,共5页Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金重点项目(30630048)

摘　　要：根据H9亚型禽流感病毒HA基因上的保守序列,设计合成引物,以阳性H9亚型禽流感病毒HA基因重组质粒为标准品做标准曲线,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0.997,灵敏度约为6拷贝/μL,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,对193份临床泄殖腔棉拭样品的检测,其结果与经典病毒分离方法符合率大于90.0%。该方法为H9亚型禽流感病毒检测提供了一种特异、敏感、快速、较便宜、高通量、生物安全性好的定量检测手段,将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良好的应用前景。According to the conservative region of HA gene of H9 subtype avian influenza virus （ AIV）, a pair of primers was designed and synthesized. A serial of ten fold dilutions of positive plasmid was prepared and used as standard. The standard curve revealed the good linear relationship between Ct （cycle threshold） and template concentration （R2 =0. 997 ）. No cross-reaction was detected against other avian viruses. The detection limit was 6 copies of AIV genome. The coincidence rate was more than 90. 0% with the traditional virus isolation method in detecting 193 clinical cloaeal swab samples. It suggested that a highly specific and sensitive Real-time RT-PCR method for detection of H9 subtype AIV had been established in this study. This method has good prospects in the rapid and quantative detection of AIV H9 subtype in clinical samples.

关 键 词：禽流感病毒 H9亚型 荧光定量RT-PCR 

分 类 号：S855.3[农业科学—临床兽医学]