Development of a SYBR Green I real-time PCR assay for detection of Mycoplasma gallisepticum

To develop a specific assay for quantitative detection of Mycoplasma gallisepticum,a SYBR Green I real-time PCR assay was established with a pair of primers targeting M.gallisepticum 16 S rDNA gene.A recombinant plasmid of pMD-16 S containing 186 bp of 16 S rDNA target fragment was constructed as a standard template to set up the standard curve which exhibited a dynamic linear ranging from 1.96×10~8 copies/μL to 1.96×10~2 copies/μL.Then,the specific and sensitive tests showed that real-time PCR assay was only detected M.gallisepticum with the detection limit of 1.96×10~1 copies/μL,but had no cross-reaction with other avian respiratory tract pathogens examined.The variations of both inter- and intra-assay were less than2%.Moreover,thirty clinical samples were examined for M.gallisepticum,the positive rate was 40%by real-time PCR,but it was only 33%and 23%with conventional PCR and serum plate agglutination test,respectively,showing that this assay was much more sensitive than conventional detection assays.The results showed that this real-time PCR assay provides an effective method for quantitative detection and vaccine quality control of M.gallisepticum.