The Development Of Real Time PCR Assay For Detection Of Mycoplasma Hyopneumoniae

Mycoplasma hyopneumoniae (Mhp) is the pathogen of Swine Enzootic Pneumoniae (EP). The classical methods for quantitative detection of Mhp are CCU(color change unit), CFU(colony forming unit), Microscopic Counting or PCR assay with labor-intensive, time-consuming and indefinite results.Recently, Real Time PCR has been used for detection and identification of mycoplasma, and two of them are about qualitative detection of Mhp, which still could not solve its problem about quantitative detection.In this research, primers were designed according to the p110 gene, p97 gene and p46 gene of Mhp 232 strain (GENBANK ACCESSION No. NC₀06360) as well as the p76 gene, p97 gene and p46 gene of Mhp J strain (ATCC 25934, GENBANK ACCESSION No. NC₀07295) and Mhp 7448 strain (GENBANK ACCESSION No. NC₀07332). After analyzing the amplifying curve,standard curve and melting curve of the real time PCR to find out the optimal primers and constructing standard plasmid containing the p110 gene of Mhp, the SYBR GREEN quantitative Real Time PCR based on p110 (p76) gene has been established, which is precise, quick, sensitive, specific, repeatable and contamination-free and can detect 10 copies of standard plasmid DNAperμL. This method can be used for quantitative detection of Mhp in the culture medium or fermented for vaccine production.