Abstract: The 128 bp specific and consensus sequence of Eastern equine encephalitis virus (EEEV) was amplified by RT-PCR and cloned into pMD20-T vector,and conducted in vitro transcription to prepare standard cRNA.10 fold serial diluted cRNA were used as standard templates for RT-PCR to quantify the genomic copy number of EEEV.We developed a TaqMan MGB Real-time RT-PCR to detect EEEV.Sensitivity assay result showed that the established TaqMan MGB Real-time RT-PCR could detect 10 copies/μL cRNA.The specificity assay exhibited that negative control and the other equine pathogens (EHV-1,EAV,EIV H3N8,WNV,WEEV and JEV) could not be detected.A good linear correlation was demonstrated in the standard curve for TaqMan MGB Real-time RT-PCR within the range of 1.0×10¹ to 1.0×10⁶ copies/μL with a correlation coefficient of 0.998 and a standard curve of y=40-3.35logx.The results demonstrated that TaqMan MGB Real-time RT-PCR was 100 fold more sensitive than conventional RT-PCR.The results suggested that the convenient,specific,high performance and sensitive method of TaqMan MGB Real-time RT-PCR for the detection of EEEV was successfully established in this study.

Key words: Eastern equine encephalitis virus (EEEV); TaqMan MGB; Real-time RT-PCR