嗜肺军团菌实时荧光定量PCR快速检测方法的建立

Development of a Rapid TaqMan Real-Time PCR Assay for Detection of Legionella pneumophila

目的:建立针对嗜肺军团菌Mip基因的实时荧光定量TaqMan PCR检测方法,并进行自来水和空调冷却水模拟标本的检测评价。方法:根据嗜肺军团菌Mip基因的特异性序列设计引物和TaqMan探针,建立嗜肺军团菌的实时荧光定量TaqMan PCR快速检测方法,对方法进行灵敏度及特异性评价,并对自来水和空调冷却水模拟标本中的嗜肺军团菌进行检测。结果:建立的方法对嗜肺军团菌的检测具有高度特异性,与3种非嗜肺军团菌和6种其他呼吸道病原均没有交叉反应;基因组DNA的检测灵敏度为1.6 pg/μL,模拟自来水和空调冷却水标本的检测灵敏度为10CFU/mL。结论:建立的TaqMan荧光定量PCR方法特异、灵敏、快速,适于嗜肺军团菌的日常监测和暴发疫情的应急诊断。

Objective： To develop a TaqMan-based real-time PCR method for rapid detection of LegioneUa pneumophila. Methods： The sequence of maerophage infectivity potentiator（Mip） gene was downloaded from GenBank and the specific primers and TaqMan probe were designed in the conserved region of the Mip gene for L.pneumophila. Then, the real-time PCR array for rapid detection of L.pneumophila was developed and its speeificity and sensitivity were evaluated. Simulated environment water samples were used to assess the assay. Results： Only L. pneumophila strains generated fluorescent signals, and no cross-reaction was observed for the differential control strains including three non-pneumophila strains and six other respiratory pathogens. The detection limits were 1.6 pg/μL with genomic DNA of L.pneumophila, and 10 CFU/mL with simulated water samples. Conclusion： The TaqMan real-time PCR assay described here is specific, sensitive and rapid for detection of L.pneumophila, and this assay could be used for laboratory-based monitoring and emergency detection of L.pneumophila.