Research Of Optimizing The Growth Conditions Of Mycoplasma Pneumoniae By Using TaqMan Real-time Fluorescence Quantitative PCR Method

Objective:The study is dedicated to optimizing a prompt real-time quantitation PCR（qPCR）method by amplification with species specific 16S ribosomal RNA of Mycoplasma pneumoniae to quickly detect the M.pneumonia,so as to facilitate clinical diagnosis and optimize the culture conditions of M.pneumonia in scientific research and explore the potential factors that affect the growth of M.pneumonia.Methods:1.By comparing the M.pneumonia gene sequence in Gen Bank,selecting conserved regions and applying Primer 5.0 soft design specific primers and probes;2.Use the recombinant plasmid as an absolute quantitative template to establish a standard curve,and construct an optimized M.pneumonia TaqMan qPCR detection method;3.Apply the optimized M.pneumonia qPCR method to diagnose the clinical specimens,and analyze the clinical practicability of this method;4.Optimized the sampling method of M.pneumoniae and the processing method of M.pneumoniae qPCR sample;5.Comparison of M.pneumoniae qPCR detection method with M.pneumoniae CCU method;6.Optimized M.pneumoniae culture in terms of glucose concentration,FBS concentration,human serum antibodies,different drugs,different cells and media,and explored the effects of p H of the medium,repeated freezing and thawing,and shaking culture on the growth of M.pneumoniae.Results:1.The results showed that the linearity of standard curve was good:R²=1.000,Slope=-3.7062,E=86.1%;2.This method can only amplify the M.pneumonia positive control,and the other 9 common respiratory pathogens have no crossover with negative results,indicating that the method has strong detection specificity;3.The minimum detection limit of this method is 10¹copis/μL,the maximum detection limit is 10¹⁰copis/μL,indicating that the method has high detection sensitivity;4.The coefficient of variation for within-group and between-group of this method is less than 2%,indicating that the method has good reproducibility;5.Comparing the results of 45 clinical specimens with commercial M.pneumonia DNA detection kits or our method,the positive rate of the M.pneumonia qPCR method established in this experiment was 91.1%,which was slightly higher than the positive rate of the commercial M.pneumonia DNA detection kits.6.The M.pneumonia concentration detected by the Vortex oscillation sampling or metal bath high-temperature processing sample was the highest;7.The M.pneumoniae qPCR detection method was faster and more accurate than the M.pneumoniae CCU method;8.The 1.0%glucose group,30%FBS group,Buchenned,co-cultured with THP-1 cells could make the M.pneumonia grow faster,healthy people’s serum and most drugs inhibited the growth of M.pneumonia;9.With the growth of M.pneumonia,the p H value of the culture medium also decreased continuously,and while the M.pneumonia DNA concentration reached the peak,the p H value also droped significantly,but the p H value has no significant effect on the M.pneumonia qPCR detection;10.Repeated freezing and thawing reduced the growth activity of M.pneumonia;11.M.pneumonia conventional culture was better than M.pneumonia shaking culture.Conclusion:1.The TaqMan qPCR detection method for the M.pneumonia had quicker reation and more evident specificity,higher sensitivity and better repetitiveness than the regular PCR.It provides a practical method for rapid detection of M.pneumonia infection in clinical and epidemiological investigation.2.The established M.pneumonia qPCR method could be used in M.pneumonia growth detection and optimization of culture conditions.