西方马脑炎病毒TaqMan MGB荧光定量RT-PCR检测方法的建立  

Development of TaqMan MGB Real-time PCR for Detection of Western Equine Encephalitis Virus

作　　者：郑小龙[1] 王群[1] 邓明俊[1] 张晓文[1] 孙明君[1] 朱来华[1] 

机构地区：[1]山东出入境检验检疫局,山东青岛266002

出　　处：《动物医学进展》2016年第1期6-10,共5页Progress In Veterinary Medicine

基　　金：国家质检总局科技项目(2014IK240)

摘　　要：通过RT-PCR从西方马脑炎病毒(WEEV)中扩增得到1 317bp特异的保守序列,将其克隆到pMD-20T载体中,进行体外转录,制备标准品cRNA。以10倍比稀释的cRNA为模板,进行TaqMan MGB荧光定量RT-PCR扩增并制作标准曲线,建立了WEEV荧光定量RT-PCR检测方法。对建立的方法进行了特异性和敏感性试验。结果表明,建立的荧光定量RT-PCR方法最低可检测10拷贝的cRNA;且与马鼻肺炎病毒(EHV-1)、马动脉炎病毒(EAV)、马流感病毒(EIV,H3N8)、西尼罗病毒(WNV)、东方马脑炎病毒(EEEV)和日本脑炎病毒(JEV)不发生交叉反应。与常规RT-PCR相比,该方法更加快速,特异性和敏感性更高,灵敏度为常规RT-PCR方法的100倍。The 1 317 bp specific and consensus sequence of Western equine encephalitis virus（WEEV）was amplified by RT-PCR and cloned into plasmid pMD-20 T,RNA was transcribed in vitro using linearised plasmid DNA.Serial dilutions of cRNA were used as standard templates for real-time RT-PCR to quantify the genomic copy number of WEEV.We developed a TaqMan MGB real-time RT-PCR to detect WEEV.Sensitivity analysis showed that the developed TaqMan MGB real-time RT-PCR could detect 10copies/μL.The specificity assay exhibited that negative control and the other equine pathogens could not be detected.The result demonstrated a 100 fold sensitivity of the real-time RT-PCR assay was developed compared with gel-based real-time RT-PCR method.

关 键 词：西方马脑炎病毒 TAQMAN MGB 荧光定量RT-PCR 

分 类 号：S855.3[农业科学—临床兽医学]