[Establishment and application of loop-mediated isothermal amplification method for rapid detection]

Abstract

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 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.

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.