Standardization of a Real-time PCR System for Quantitative Detection of Mycoplasma hyopneumoniae

This study was conducted to develop a method for accurate quantification of Mycoplasma hyopneumoniae during vaccine production or experimental research. Primer and probe concentration that gave the highest ΔRn and the lowest Ct were selected to establish the real-time PCR system for the detection of M. hyopneumoniae. Template DNA of M. hyopneumoniae was extracted by boiling under different conditions and detected by real-time PCR to determine the optimal conditions for DNA extraction. Thereafter, intra-and inter-batch reproducibility tests were carried out using a standard plasmid to evaluate the stability of the PCR system. Subsequently, the effect of medium composition on the quantitative detection was evaluated. Finally, the correlation between real-time PCR and CCU method was explored. The optimal primer and probe concentration for real-time PCR were 0.4 and 0.2 μmol/L, respectively. The intra-and inter-batch coefficients of variation(CV) in Ct value of 10～4-10～9 copies/μl standard plasmid were <5%, indicating good reproducibility of the real-time PCR system. Following incubation in a boiling water bath for 10 min, M. hyopneumoniae samples can be used directly as a template in subsequent real-time PCR assays,and good intra-batch and inter-batch reproducibility was observed. The working concentration of KM2 medium should be less than the 1/10 of the concentration of the stock solution to minimize its influence on the quantitative detection. Spearman's correlation analysis revealed that the log of CCU and the log of DNA copy number had a significant positive relationship(r=0.797,P=0.000). Thus, the two methods can be used in combination in the quantitative detection of M. hyopneumoniae. In summary, a rapid, stable and accurate quantitative PCR system for detecting M. hyopneumoniae culture was established in this study, which provides a technical means for accurate quantification of M. hyopneumoniae in vaccine production and laboratory tests.