The conserved region was used to design and synthetize primers and TaqMan probe by aligning nucleic acid sequence of representative strains of western equine encephalomyelitis virus (WEEV). A real-time RT-PCR(RRT-PCR) was developed to detect WEEV by the optimization of primers, probe and reaction conditions. The established RRT-PCR was used to detect a panel of extracted virus RNA/DNAs including those of WEEV, eastern equine encephalomyelitis virus(EEEV), equine arteritis virus(EAV), equine herpes virus type 1(EHV-1), equine influenza virus subtype H3N8(EIV H3N8) with only positive result for WEEV RNA, but negative for other four virus DNA/RNAs. Furthermore, cRNA(7442nt~10011nt) of WEEV McMillan strain was prepared by in vitro transcription. After copies calculation, dilution, aliquot, homogeneity and stability testing, the cRNAs were used to evaluate the established RRT-PCR. The result indicated that the detection limit of RRT-PCR was 10 copies per reaction using in vitro transcribed cRNA, It only took 4h to detect 197 blood samples from imported horses, suggesting that the RRT-PCR was a rapid, sensitive and specific method, and the method can be used as a technical reserve for WEEV screening of imported horses.%选取西部马脑炎病毒代表株进行序列比对分析，选择保守区域，设计合成引物和TaqMan探针。经对反应体系和条件进行优化，建立了检测西部马脑炎病毒的实时荧光RT-PCR检测方法，应用建立的方法对西部马脑炎病毒核酸、东部马脑炎病毒核酸、马动脉炎病毒核酸、马疱疹病毒1型核酸、马流感病毒H3N8亚型核酸进行检测，结果表明建立的方法只能检出西部马脑炎病毒核酸，与另外4种病毒核酸无交叉反应。进一步通过体外转录制备了西部马脑炎病毒McMillan株7442~10011的cRNA片段，经拷贝数计算、稀释、分装、均匀性和稳定性检验后，作为质控品对建立的方法进行评价。结果显示，所建立的方法其分析灵敏度可达10拷贝。对197份进口马血样品检测用时仅4小时，表明该检测技术快速、灵敏、特异。因此，本研究建立的方法可作为技术储备用于进口马匹的疫病筛查。
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