嗜肺军团菌荧光定量PCR检测

Detection of Legionella pneumophila by using fluorescence quantitative PCR

目的建立特异、快速、敏感的TaqMan探针荧光定量PCR方法,用于检测嗜肺军团菌mip基因。方法嗜肺军团菌及其他军团菌DNA模板来自中国疾病预防控制中心呼吸道室,嗜肺军团菌地方株及其他对照株由本中心菌种室供给。利用嗜肺军团菌mip基因保守序列设计引物和TaqMan探针,对其进行筛选,同时对荧光定量PCR反应条件和反应体系进行优化,并对嗜肺军团菌、非嗜肺军团菌及其他细菌进行检测,验证该方法的特异性、敏感性、重复性等。结果该方法在检测多株嗜肺军团菌时,均出现阳性信号,而对非嗜肺军团菌和其他细菌则未有阳性扩增结果出现。检测灵敏度达10个拷贝/反应,从菌株核酸的提取至检测完成仅需2 h左右,可减少常规PCR操作的污染机会。结论TaqMan荧光定量PCR方法可定量检测嗜肺军团菌,具有特异性高、快速、敏感等特点,适于外环境污染源状况的调查及应急事件的快速定量检测。

Objective To develop a specific, rapid and sensitive fluorescence quantitative PCR assay for detection of Legionella pneurnophila. Methods The genomic DNA of Legionella pneurnophila and Legionella spp was from Chinese Center for Disease Control and Prevention, and then the local strains of Legionella pneurnophila and other control strains were obtained from Zhejiang Provincial Center for Disease Control and Prevention. The primers and TaqMan probe were designed in conservative sequence of Legionella pneumophila mip gene, and then the PCR condition and the reaction system were optimized simultaneously. The specificity, sensitivity, reproducibility of the assay were evaluated. Results This assay had high sensitivity and specificity for detecting L. pneurnophila but not to the other Legionella spp. and other strains, the sensitivity of this assay was 10 copies/reaction. The whole process from extraction of genomic DNA of strains to assessment of the results could be done in about 2 hours. It was a simple, convenient assay with good reproducibility. Furthermore, this method can also decrease the probability of contamination. Conclusion The fluorescence quantitative PCR provides a specific, rapid and sensitive method for quantitative detection of L. pneurnophila. It is helpful for the rapid detection of environment source of pollution, emergency and clinical samples.