Zhu Shui-rong, Wang Zhi-gang, Zhang Zheng, Lu Yi-yu, Mei Ling-ling, Zhan Li
The Institute of Microbiology, Zhejiang Center for Disease Control and Prevention, Hangzhou 310051, China.
Zhonghua Liu Xing Bing Xue Za Zhi. 2009 May;30(5):481-5.
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.
Five primers (2 Inner Primers, 2 Outer Primers and a Loop Primer) for the LAMP test were designed by targeting the mip 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.
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.
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.
开发一种环介导等温扩增（LAMP）方法，用于病原体检测部门（PDD）或小型实验室中快速诊断嗜肺军团菌。
针对嗜肺军团菌的mip基因设计了5条用于LAMP检测的引物（2条内引物、2条外引物和1条环引物），并优化了LAMP反应体系。对12株嗜肺军团菌、45株本地菌株、6株非嗜肺军团菌菌株、11株其他菌株和59份环境水样进行分析，以评估LAMP扩增的特异性和敏感性。同时，还将LAMP的结果与生化培养和定量PCR方法的结果进行了比较。
嗜肺军团菌的扩增产物肉眼观察呈绿色，在凝胶上呈梯状条带，而非嗜肺军团菌及其他菌株的产物肉眼观察仍为橙色，在凝胶上无条带。LAMP的检测率高于生化培养和实时PCR方法。LAMP方法的反应时间仅为1.5小时，检测限为5 cfu/反应。此外，LAMP结果仅通过肉眼观察即可确定。
针对嗜肺军团菌mip基因的LAMP检测方法对嗜肺军团菌的检测具有快速、特异、灵敏的特点。该方法不仅减少了对复杂设备的依赖，而且有在PDD、小型实验室、应急机动车或现场调查中更广泛应用的潜力。
