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目的建立敏感、特异的Taq Man-MGB探针实时荧光定量-聚合酶链反应(real-time PCR)快速检测方法,为临床标本中肺炎支原体快速检测提供技术支持。方法选取肺炎支原体重复序列Rep MP23中保守区域设计、合成特异性扩增引物和Taq Man-MGB探针,建立并优化real-time PCR检测方法。对优化后的方法使用标准浓度核酸进行扩增效率、灵敏度及特异度评价,并与已报道的肺炎支原体荧光PCR方法进行比较。结果本研究建立的Taq Man-MGB探针荧光PCR方法对肺炎支原体的检测限为0.2 cfu,比普通Taq Man real-time PCR检测限(3~5 cfu)提高了一个数量级。其检测标准曲线的循环阈值(Ct)与模板拷贝数呈良好的线性关系(R2=0.999)。用该方法扩增23种呼吸道常见病原菌染色体及人类染色体,结果均为阴性,特异度为100%。结论 Taq Man-MGB探针real-time PCR方法可快速、灵敏、特异地检测肺炎支原体,有很好的应用前景与价值。
Objective To establish a sensitive and specific Taq Man-MGB probe for rapid detection of Mycoplasma pneumoniae by real-time fluorescence quantitative-polymerase chain reaction (real-time PCR). Methods The conserved region of Mycoplasma pneumoniae repeats Rep MP23 was selected and specific primers and Taq Man-MGB probes were synthesized. The real-time PCR method was established and optimized. Amplification efficiency, sensitivity and specificity were evaluated using the standard concentration of nucleic acids for the optimized method and compared with the reported Mycoplasma pneumoniae fluorescence PCR method. Results The detection limit of Mycoplasma pneumoniae by Taq Man-MGB probe fluorescence PCR method was 0.2 cfu, which was an order of magnitude higher than the detection limit of normal Taq Man real-time PCR (3 ~ 5 cfu). The cycle threshold value (Ct) of the standard curve of the detection showed a good linear relationship with the template copy number (R2 = 0.999). This method was used to amplify 23 common pathogen chromosomes of respiratory tract and human chromosomes. The results were negative and the specificity was 100%. Conclusion The TaqMan-MGB probe real-time PCR method can detect Mycoplasma pneumoniae rapidly, sensitively and specifically, and has good application prospect and value.