嗜肺军团菌荧光定量PCR方法的建立及在公共场所集中空调检测中的应用

Establishment and application of fluorescence quantitative PCR to detecting Legionella pneumophila in central air conditioning water systems in public places

目的建立特异、敏感、快速的嗜肺军团菌荧光定量PCR检测方法。方法使用Beacon Designer软件针对嗜肺军团菌mip基因设计特异性引物与探针,验证所建方法的特异性、敏感性及重复性,并对公共场所的真实环境样本进行检测。结果该法特异性好,仅嗜肺军团菌出现扩增曲线;检测灵敏度达到6. 60 pg/μl,为普通PCR的10倍; 69份水样中检测出16份阳性,阳性率为23. 2%。结论公共场所集中空调系统存在一定的嗜肺军团菌污染,有发生军团菌病的风险。该法快速且具有良好的特异性、敏感性和重复性,适用于公共环境中嗜肺军团菌污染调查及快速检测。

Objective To establish a specific, sensitive and quick test method for the detection of Legionella pneumophila fluorescence quantitative PCR. Methods The specific primers and probes for L. pneumophila mip gene were designed using Beacon Designer software so as to verify the specificity, sensitivity, and repeatability of the established method. Real environmental samples in public places were also tested. Results The specificity of the method was good and there was a positive amplification curve for L. pneumophila, but none for other bacteria. The detection sensitivity was 6.60 pg/μl, which was 10 times that of ordinary PCR. Sixteen of the 69 water samples were positive and the positive rate was 23.2%. Conclusion The central air conditioning system in public places has been polluted to some extent by L. pneumophila. So that there is a risk of legionellosis. This method is quick and has better specificity, sensitivity and repeatability. It is applicable to investigation and rapid detection of L. pneumophila contamination in public places.