| Mycoplasma bovis disease with a Variety of disorders is a kind of infectious disease. caused by the Mycoplasma bovis.For establishing fast and accurate diagnosis method of the disease,primers and probes were designed and synthesized selecting OPPD/F as a specific gene.The TaqMan real-time Fluorescent Quantitation PCR detection method for the Mycoplasma bovis was established on the basis of the reference literature.Objective:The study was aimed at construction of a prompt TaqMan real-time fluorescent quantitation PCR method for the detection of Mycoplasma bovis. Methods: According to the gene sequence of the Mycoplasma bovis OPPD/F included in the Genbank (accession number:AF130119), the Primer 5.0 software was used to design specific primer and TaqMan probe and the TaqMan real-time Fluorescent Quantitation PCR detection method for the Mycoplasma bovis was established using recombinant plasmid as the template of absolute quantitation. Results:It was showed the TaqMan real-time Fluorescent Quantitation PCR detection method was well linear relationship, with the correlation coefficient R2=0.994 and the amplification efficiency E=1.28 of the related standard curve. The sensitivity of the TaqMan real-time fluorescent quantitation PCR method was 10 times higher than that of the regular PCR method. It was identified that the TaqMan real-time fluorescent quantitation PCR method had quite well specificity through the negative results of the detection for 9 kinds of bacteria and viruses. In addition, the detection Ct values within and between groups were all lower than 2%, which demonstrated that the method had well repetitiveness. Conclusion:The TaqMan real-time fluorescent quantitation PCR detection method for the Mycoplasma bovis had quicker reaction and more evident specificityã€higher sensitivity and better repetitiveness than the regular PCR. |
