Development of a TaqMan real-time PCR for detection of the Mycoplasma mycoidessubsp. capri

Abstract

BackgroundThe purpose of this study was to develop a real-time quantitative fluorescence polymerase chain reaction (QF-PCR) method for the rapid detection of Mycoplasma mycoidessubsp. Capri (Mmc). Based on the Mmc MLC_1770 gene sequence (No.FQ377874.1) from GenBank, specific primers and a TaqMan probe were designed using Beacon Designer 7.9. The recombinant plasmid of Mmc-1770 was constructed as a positive control for the construction of the standard curve and assessment of the specificity, sensitivity, and repeatability of the method. ResultsThe results indicated that a sensitivity of 11copies/μL could be achieved with this detection method, which is 1000 times higher than that of conventional PCR. Cross-reactions with Mycoplasma capricolum subsp. capri pneumoniae (Mccp), Mycoplasma leachii (M. leachii), Mycoplasma agalactiae (M. agalactiae), Mycoplasma capricolum subsp. capricolum (Mcc), Escherichia coli(E. coli), Pasteurella multocida (Pm), Staphylococcus ureus (SA), Mannheimia haemolytica (Mh), Mycoplasma arginine (M. arginini), Mycoplasma ovipneumoniae (Mo), Mycoplasma bovis (M. bovis), and orf virus (ORFV) were not observed. The intra- and inter-assay coefficients of variation were 0.60–1.52% and 0.49–0.78%, respectively, which indicated the method was highly repeatable. Subsequently, the method was used to test 188 suspected Mmc-infected samples, and Mmc was detected in 27, giving a positive rate of 14.36%. ConclusionThe results of this study demonstrate that the developed QF-PCR method has high sensitivity, specificity, and repeatability, and provides reliable technological support for the detection, prevention, and control of Mmc infection.