嗜肺军团菌环介导等温扩增快速检测方法的建立与应用

Establishment and application of loop-mediated isothermal amplification method for rapid detection of Legionella pneumophila

目的建立一种适合基层检验部门及小型实验室使用的快速检测嗜肺军团菌的方法。方法应用环介导等温扩增技术（LAMP），针对嗜肺军团菌mip基因设计5条引物（2条内引物、2条外引物及1条环引物），并对LAMP反应条件和反应体系进行优化。为验证该方法的特异性、敏感性，针对种系背景明确的12株嗜肺军团菌实验对照菌株、45株嗜肺军团菌地方分离株、6株非嗜肺军团菌、11株其他细菌以及59份外环境水样本进行检测，并将其结果与传统培养分离方法及定量PCR方法比较。结果所检测的菌株样本中不同血清型嗜肺军团菌经LAMP检测均呈绿色为阳性，非嗜肺军团菌及其他菌株检测均呈橙色为阴性。实验结果表明，LAMP方法的检出率高于传统培养分离方法及定量PCR。应用该方法从菌株核酸的提取至检测完成仅需1．5h左右，该体系检测灵敏度为5cfu／ml，而且其检测结果在日（灯）光下通过肉眼即可判断。结论实验建立的LAMP方法能够快速、灵敏、特异地检测嗜肺军团菌，适合基层检验部门及小型实验室与现场监测等使用。

Objective To develop a loop-mediated isothermal amplification （LAMP） method for rapid diagnosing of Legionella pneumophila in the Pathogen Detection Department（PDD） or in small-scale laboratory. Methods Five primers （2 Inner Primers, 2 Outer Primers and a Loop Primer） for the LAMP test were designed by targeting the rnip gene of L.pneumophila and reaction system of LAMP reaction was optimized. 12 strains of L.pneumophila, 45 local strains, 6 non-L.pneumophila strains, 11 other strains and 59 environmental water samples were analyzed to evaluate the specificity and sensibility of the LAMP amplification. At the same time, the results of the LAMP were also compared with biochemical culture and quantitative PCR methods. Results The amplification products of L.pneumophila turned green by visual inspection and had ladder-like pattern on the gel, but non-L.pneumophila and other products from the strains remained orange by visual examination and had no band on the gel. The detection rate of LAMP was higher than the biochemical culture and the real-time PCR methods. Reaction time of the LAMP method was only 1.5 h and the detection limit of LAMP assay was 5 cfu/reaction. In addition, the LAMP results could be determined only by visual inspection. Conclusion LAMP assay targeting the mip gene of L.pneumophila appeared to be rapid, specific, and sensitive for the detection of L.pneumophila. This method not only reduced the dependence of complicated equipment but also had a potential for wider use in the PDD, small-scale laboratory, emergency motor vehicle or for field survey.