实时荧光定量-聚合酶链反应方法检测肺炎支原体
A real-time PCR assay for detection of Mycoplasma pneumoniae
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摘要: 目的 建立一种快速、灵敏、特异的肺炎支原体实时荧光定量-聚合酶链反应(real-time PCR)检测方法,以期用于临床肺炎支原体感染检测。 方法 通过测序分析和序列比对,选取肺炎支原体p1基因中保守区域设计特异性引物和荧光探针,建立和完善此real-time PCR检测方法,并进行扩增效率、灵敏度及特异度评价。与已报道的肺炎支原体常规聚合酶链反应(PCR)方法进行150份临床标本检测能力比较。 结果 建立的real-time PCR方法对肺炎支原体的检测限约为10 cfu。使用该方法对9株肺炎支原体ATCC标准株和30株临床分离株核酸扩增均为阳性;对10种其他支原体、13种常见呼吸道病原菌染色体及人类染色体扩增结果均为阴性。同时,临床标本的检测结果显示该方法检测灵敏度优于常规PCR。 结论 本研究建立的real-time PCR方法可快速、灵敏、特异地检测标本中肺炎支原体核酸,可适用于临床肺炎支原体诊断。Abstract: Objective To established a simple, rapid real-time PCR assay to detect Mycoplasma pneumoniae in clinical specimens. Methods By analyzing the p1 gene sequence of M. pneumoniae isolates, an optimized real-time PCR assay was designed. The specificity and sensitivity of this assay were evaluated and compared with conventional PCR assay using clinical specimens. Results The detection limit of the real-time PCR assay was about 10 cfu. The sensitivity and specificity of the assay were high. This real-time PCR assay was superior to conventional PCR in clinical specimen detection. Conclusion The real-time PCR assay is suitable for the detection of M. pneumoniae in clinical specimens.
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