嗜肺军团菌荧光定量PCR检测方法的建立

Detection of legionella pneumophila by fluorescence quantitative PCR method

目的:建立嗜肺军团菌mip基因荧光定量PCR检测方法。方法:利用Primer Express软件设计特异性引物和探针,对质粒标准品、嗜肺军团菌、非嗜肺军团菌及其他菌株进行检测,验证方法的特异性、敏感性和重复性,最后采集环境样本进行检测。结果:该方法特异性好,嗜肺军团菌呈现阳性结果,而非嗜肺军团菌及其他菌株均为阴性结果。检测灵敏度达293copies/μl;重复性较好,Ct值变异系数较小;检测的12份环境样本中5份阳性。结论:该方法快速且具有较好的特异性、敏感性、重复性,适于外环境嗜肺军团菌污染调查及应急事件的快速检测。

Objective： To establish a fluorescence quantitative PCR assay to detection Legionella pneumophila. Methods： Primers and probe were designed by Primer software, and plasmid standard, legionella pneumophila, non--legionella pneumophila and other strains were used to evaluate the specificity, sensitivity and repeatability of the assay. Environmental samples were collected and detected by this assay. Results： This assay had high specificity for detecting Legionella pneumophila but not to non-legionella pneumophila and other strains. The sensitivity of this assay was 293 copies/μl. The repeatability of this assay was high, the coes{fieient of variation of Ct values was low. Five of twelves environmental samples were positive. Conclusion：The fluorescence quantitative PCR provides a specific , rapid and sensitive method for quantitative detection of Legionella pnenmophila. It is helpful for the rapid detection of environment source of I.egionella pollution and emergency.