荧光环介导恒温扩增技术检测嗜肺军团菌方法的建立

目的探讨利用环介导恒温扩增技术(LAMP)建立可以准确快速检测嗜肺军团菌的方法。方法选取嗜肺军团菌、金黄色葡萄球菌、铜绿假单胞杆菌、大肠埃希氏菌、阪崎肠杆菌、单核细胞增生李斯特氏菌、鼠伤寒沙门氏菌、福氏志贺氏菌、副溶血性弧菌菌株,根据嗜肺军团菌毒力mip基因的6个特殊区域,使用Primer Explorer Version 4软件设计LAMP引物(mip-1、mip-2、mip-3),提取病原菌基因组DNA进行LAMP,评价其特异性、最低检测限和稳定性。结果筛选出mip-3引物在测定嗜肺军团菌的LAMP反应体系中扩增约10min出峰且峰值较高;非嗜肺军团菌菌株没有扩增反应;LAMP检测限可达100fg/μL;mip-3引物在10次重复检测中几乎同时出峰,稳定性良好。结论建立的LAMP检测方法具有特异性强、稳定性高特点,能快速、准确检测出嗜肺军团菌,具有较大的推广及应用前景。

Establishment of method to detect legionella pneumophila by LAMP technology

Objective To establish a accurate and rapid method of loop-mediated isothermal amplification(LAMP) for detecting Legionella Pneumophila(LP) .Methods The strains of LP ,Staphylococcus aureus ,Pseudomonas aeruginosa ,Escherichia coli ,Enterobacter sakazakii ,Listeria monocytogenes ,Salmonella typhimurium ,shigella flexneri and Vibrio parahaemolyticus were selected . According to six special domains of toxicity-related mip gene on LP ,the LAMP primers(mip-1 ,mip-2 ,mip-3) were designed by using the Primer Explorer Version 4 .0 .The genomic DNA was extracted for conducting LAMP .Then its specificity ,lowest detectable limit and stability were evaluated .Results The screened primer mip-3 appeared the peak after amplification for about 10 min in the LAMP reaction system for detecting LP ,moreover the peak value was higher ;while the strains of non-LP had no amplification reaction ;the LAMP detection limit could reach 100 fg/μL .The primer mip-3 appeared the peak almost at the same time in 20 times of duplicated detection ,and its stability was good .Conclusion The established LAMP detection method has the advantages of strong specificity and high stability ,can rapidly and accurately detect LP and has large prospect of promotion and application .