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 vac- cine production or experimental research. Primer and probe concentration that gave the highest ARn and the lowest Ct were se- lected 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 ex- traction. 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 umol/L, respectively. The intra- and inter-batch coefficients of variation （CV） in Ct value of 104-10g copies/iJI standard plasmid were 〈5%, indicating good reproducibility of the real-time PCR system. Following incubation in a boil- ing 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 corre- lation 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 Drovides a technical means for accurate quantification of M. hyopneumoniae in vaccine production and laboratory tests.