Taqman-MGB双重探针PCR技术检测军团菌

Duplex fluorescence quantitative PCR for detection of Legionella by using MGB fluorescence probe

目的建立同时检测军团菌属和嗜肺军团菌的双重荧光定量PCR方法。方法利用军团菌16SrDNA和rnip基因的特异性序列设计引物和MGB探针，两条探针5’端分别标记FAM和HEX，3’端标记MGB。优化了荧光定量PCR反应条件和反应体系，并对军团菌、嗜肺军团菌、非嗜肺军团菌及其它细菌进行检测，验证该方法的特异性、敏感性、重复性。结果该方法所检测菌株中军团茵属结果均为16SrDNA阳性（LP12、LP13除外）；嗜肺军团菌均为mip阳性，非嗜肺军团菌属除L．micdadei外均为阴性；而非军团菌菌株16SrDNA和mip检测结果均为阴性。检测灵敏度达10个拷贝／反应；重复性实验中，变异系数为0．2％～O．6％；从菌株核酸的提取至检测完成仅需2h左右。结论本文建立的TaqMan-MGB双重探针荧光定量PCR是一种定量检测军团菌的特异、快速、敏感的方法，可区分嗜肺与非嗜肺军团菌属，该方法的建立将有益于今后开展对外环境污染源进行初步卫生学调查与评估，以及应急临床标本的快速定量检测。

To develop a duplex fluorescence quantitative PCR for detecting Legionella, based on TaqMan hybridization probes technology,two pairs of primers and two MGB probes were designed to identify the 16S rDNA and mip gene respective- ly. The 16S rDNA probe was 5＇end labeled with FAM and 3＇end labeled with TaqMan-MGB, and then mip probe was 5＇end la beled with HEX and 3＇end labeled with TaqMan-MGB. The PCR reaction conditions were optimized. By this method of detec- tion Legionella, legionella pneumophila, norvlegionella pneumophila and other bacteria were detected. The specificity, sensi- tivity and reproducibility of the assay were evaluated. All the Legionella tested were positive for 16S rDNA gene by using this assay except LP12, LP13, while all the Legionella pneumophila and L. micdadei were positive for mip gene. The sensitivity of this assay was 10 eopies/μL. The coefficients of variation（CV） value were 0. 2%--0. 6%. and the whole process costs 2h. It is evident that Legionella can be detected by using duplex fluorescence quantitative PCR, which is specific, rapid and sensitive to detection L, pneumophila and other Legionella spp.