嗜肺军团菌环介导等温扩增检测法的建立

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

目的:建立一种快速简便的嗜肺军团菌分子检测方法。方法:运用环介导等温扩增技术(loop-mediated isothermal amplification,LAMP),设计一套引物特异性识别嗜肺军团菌毒力基因——mip基因的6个特殊区域,在恒温条件下,对8株嗜肺军团菌和5株其它细菌进行检测,并与普通PCR方法进行比对,验证该方法的特异性和灵敏度。结果:所有嗜肺军团菌扩增产物均产生肉眼可见的白色沉淀,而其它不同菌株均不产生沉淀;加入荧光染料smart green后,嗜肺军团菌扩增产物均产生强绿色荧光,其它菌株呈弱荧光。与普通PCR(检出限约为57.6 pg,)比,LAMP扩增反应的灵敏度大100倍左右,基因组DNA检出限为576 fg,阳性水样检出限为8 cfu/ml。结论:LAMP技术可在简易的实验环境中快速、特异、灵敏地检测嗜肺军团菌。

Objective： To establish a simple and rapid molecular detection for Legionella pneumophila.Methods： The loop-mediated isothermal amplification（LAMP） was applied for detection of Legionella pneumophila.A set of primers were designed to identify six special areas in mip gene of Legionella pneumophila.Genomic DNAs from 13 bacterial strains,including 8 Legionella pneumophila strains and 5 other bacterial strains were amplified by LAMP and general PCR method to evaluate the specificity and sensibility of LAMP.Results： All positive tubes produced visible white precipitation,and no precipitation was observed in others.By adding smart green fluorescent dye,all Legionella pneumophila positive tubes presented a strong green fluorescence,while others showed weak fluorescence.The detection rate of LAMP was higher than that of general PCR.The detection limits were 576fg with genomic DNA of Legionella pneumophila,and 8 cfu/mL with positive water samples.Conclusion： LAMP detection of Legionella pneumophila is an effective and low-cost method with high specificity and sensitivity requiring no special equipment.