Abstract: Objective To construct a recombinant plasmid as the standard for Theiler's murine encephalomyelitis virus (TMEV)detection by real-time quantitative PCR. Methods A specific TMEV untranslated region (UTR) fragment in size of 1014 bp was amplified by RT-PCR, The PCR product was ligated with pMD18-T vector and transformed into E.coli DH5α. Recombinant plasmid was identified by PCR and sequencing. The concentration of recombinant plasmid was analyzed through its absorption at 260 nm. Standard curve was constructed by real- time quantitative PCR with ten-fold serial dilutions of recombinant plasmid. Results The fragment of UTR was successfully cloned into pMD18-T vector. The quantitative standard curve of recombinant standard plasmids was obtained, statistics analysis indicated that there was a good linear correlation between the Ct value and the concentration gradient of standard plasmids. The regression value was over 0.99. Conclusion The standard plasmid for detecting TMEV with real-time PCR has been constructed successfully.

Key words: Theiler's murine encephalomyelitis virus (TMEV), Real-time PCR, Standard plasmid