TaqMan-MGB荧光定量RT-PCR技术快速检测H5亚型禽流感病毒  

Rapid Detection of Avian Influenza virus H5 by Real-Time RT-PCR and TaqMan-MGB probe

作　　者：卢亦愚[1] 严菊英[1] 冯燕[1] 徐昌平[1] 史雯[1] 茅海燕[1] 

机构地区：[1]浙江省疾病预防控制中心,浙江杭州310009

出　　处：《中国病毒学》2006年第5期472-476,共5页Virologica Sinica

基　　金：浙江省自然科学基金重点项目(Z303909)

摘　　要：建立以TaqMan-MGB荧光探针为特点的荧光定量RT-PCR方法,用于检测H5亚型禽流感病毒。针对H5亚型禽流感病毒血凝素(HA)基因保守区域设计特异性引物与TaqMan-MGB荧光探针,筛选并优化荧光定量RT-PCR反应体系与反应条件,用以提高方法的特异性、敏感性与准确性;并通过体外克隆技术建立病毒基因拷贝数进行定量分析。结果表明:引物与探针的优化浓度分别640nmol/L和480nmol/L,体系具有良好的保守性和特异性,与其他呼吸道病毒均无交叉反应。方法检测灵敏度为100拷贝/反应,标准曲线线性范围为107～102拷贝/反应,从病毒核酸提取至检测完成仅需3h左右,操作简便,重现性好。本研究建立的TaqMan-MGB荧光定量PCR方法特异、敏感、快速,适合于临床实验室进行H5亚型禽流感病毒的快速定量检测。A real-time reverse transcription-polymerase chain reaction （RRT-PCR） assay based on TaqMan-MGB probe was developed to rapidly detect avain influenza virus subtype H5. The assay, which was based on primers and TaqMan-MGB probes selected from highly conserved regions of the hemagglutinin gene of avain influenza virus subtype H5, was optimized in a reaction system and a condition to improve the sensitivity, specificity and accuracy. In addition, colone technology was used to develop a quantitative PCR format with virus copies amount. The results showed that the best concentration of primers and probe was 640nmol/L and 480nmol/L, respectively. None of the negative control samples showed false-positive reactions when done in duplicates. The detection limit of the assay was 100 copies per reaction. A linear standard curve was obtained between 10^2 and 10^7 DNA copies/reaction. It took only three hours from viral RNA extraction to complete the detection. The assay was simple and highly reproducible. This real-time RT-PCR assay is an excellent method suitable for rapid and quantitative detection of avian influenza virus H5 under clinical conditions.

关 键 词：荧光定量RT-PCR TAQMAN-MGB探针 H5亚型禽流感病毒(AIV H5) 

分 类 号：S854.43[农业科学—临床兽医学]