Development And Application Of A Real-Time Fluorescence Quantitative PCR Assay For Detection Of Mycoplasma Gallisepticum

Mycoplasma gallisepticum (Mycoplasma gallisepticum, MG,) also called Chicken blood mildew physique, is a major pathogen causing chronic respiratory disease of chickens, and an important pollution source of mycoplasma contamination in the production of biological products and live vaccine. So to establish an effective, strong specificity of rapid detection method has the very vital significance. Real-time fluorescent quantitative PCR technology with the characteristics of the pathogen rapid diagnosis, high specificity, strong sensitivity and accurate quantification is suitable for use in Mycoplasma gallisepticum detection and clinical diagnosis.According to MG16SrRNA, TM-1and PvpA three gene sequences published in GenBank, three pairs of primers were designed and synthesized. After amplification by PCR, the target genes were cloned into pMD19-T vector carrier, and the recombinant plasmids were constructed. Through double enzyme, PCR and sequencing identification, the constructed recombinant plasmids were identified and could be used as the standard plasmid DNA. Then the constructed recombinant plasmids as a template are for fluorescence quantitative PCR amplification detection. After the reaction conditions and reaction system was optimized and by analyzing the amplification curves, standard curve, melting curve,16SrRNA, TM-1, PvpA three gene sequences as the target sequence of MG SYBR GREEN real-time fluorescent quantitative PCR detection method are set up. The results showed that the PCR detection methods established by treating16SrRNA as the target sequence had the highest sensitivity for8.56×101copy/μL; TM-1gene for target gene took second place, the lowest detection quantity is6.01x101copy/mu L; When treating PvpA as a target gene, the detection sensitivity could up to6.01×101copy/μL. And no cross-reaction with other controls was found.In a test of120chicken embryos (allantoic fluid) and60live vaccine samples, with16SrRNA standard plasmid as the target, pollution detection rate was47.2%(85/180); TM-1standard plasmid as the target, the detection of pollution rate was39.4% (71/180); When PvpA standard plasmid as the target, the contamination rate was37.2%(67/180). This method was proved to be rapid, accurate and have good sensitivity, specificity, repeatability,which can be used for rapid quantitative detection of MG culture, tissue samples or vaccines.