TaqMan-MGB探针实时荧光定量-聚合酶链反应检测肺炎支原体方法的建立
Establishment of TaqMan-MGB real-time PCR assay to detect Mycoplasma pneumoniae
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摘要: 目的 建立敏感、特异的TaqMan-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RepMP23中保守区域设计、合成特异性扩增引物和TaqMan-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TaqMan-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TaqMan 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 TaqMan-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。Abstract: Objective To establish a sensitive and specific TaqMan-MGB real-time PCR assay to detect Mycoplasma pneumoniae from clinical specimens. Methods By analyzing the RepMP23 gene sequence of M. pneumoniae isolates, an optimized real-time PCR assay was designed. The specificity, sensitivity and amplification efficiency of this assay were evaluated with standard concentration M. pneumoniae DNA, and compared with routine TaqMan real-time PCR assay. Results The detection limitation of this TaqMan-MGB real-time PCR assay was about 0.2 cfu, about 10 times higher than routine TaqMan real-time PCR assay. The specificity of the assay seemed to be 100% in the detection of 23 common respiratory tract pathogens and human DNA. Conclusion The TaqMan-MGB real-time PCR assay was superior than TaqMan real-time PCR method. It is suitable for the detection of M. pneumoniae in clinical specimens.
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