Establishment Of Loop-mediated Isothermal Amplification Method For Detection Of Legionella Pneumophila

Background and Objective Legionella pneumophila is environmental infectious pathogens that can cause severe acute respiratory infectious diseases. It is one of the most important pathogen of atypical pneumonia. In 1976 the bacterium first appeared at a military party of the United States, so named Legionella. Legionella pneumophila mediated by the aerosol can cause epidemic outbreak or distribution. Pneumonia caused by Legionella can not be self-healing. People will be possibly die in 1-7 days if not treated in time. Mortality rate was as high as 30%, and distribution of mortality even as high as 80%. With development of social economic and living standards, more and more urban buildings have installed central air conditioning ventilation system to provide comfortable environment. However, because of the defect of the air conditioning and ventilation system, it is possible to cause air pollution, particμlarly the contamination by pathogens that may lead to respiratory tract infection. And now it has became one of the major sources of indoor air pollution. Thus, the disease caused by Legionella is a significant public health problem, which is highly concerned about.Isolation of Legionella pneumophila and serological detection sensitivity is low, with the detect rate of 25%-75% according to the relevant reports, required a longer incubation time, resulting in a lower diagnosis of Legionella disease and higher mortality rates. Current molecμlar biology techniques require expensive and sophisticated equipment and professional technical staff. However, primary laboratory can not meet these requirements. This article aims to establish a fast, easy and lower-cost molecμlar detection method for Legionella pneumophila for primary laboratory of the port. Loop-mediated isothermal amplification (loop-mediated isothermal amplification, LAMP) is a new nucleic acid amplification method developed in recent years. This method not only maintain the advantages of traditional PCR technology, but also further enhance the specificity and shorten the reaction time; in particμlar this reaction performed without the use of thermal cycle, and can be done under isothermal reaction. After the reaction, the amplification products can be observed with the naked eye. So these advantages promote the application of this technology.Methods The loop-mediated isothermal amplification (LAMP) that amplifies DNA with high specificity and rapidity under an isothermal condition was applied for rapid detection of Legionella pneumophila. A set of primers were designed specifically to identify six special areas of Legionella pneumophila's virulence gene-mip gene. Genomic DNAs from 13 bacterial strains including 18 Legionella pneumophila strains were amplified using LAMP, compared with general PCR method to evaluate the specificity and sensibility of LAMP.Resμlts The loop-mediated isothermal amplification of Legionella pneumophila completed in about 1 hour. All positive strains produce visible white precipitation, no amplicon was observed in other bacterial strains. Adding smart green fluorescent dye, all Legionella pneumophila produced a strong green fluorescence, other strains showed weak fluorescence. The detection rate of LAMP was higher than general PCR. Legionella pneumophila genomic DNA and water samples in the method detection limits were 576fg and 8cfu/ml.Conclusion In a simple experimental environment, the detection of Legionella pneumophila by LAMP technology is an effective and low-cost procedure with high specificity and sensitivity that requires no specialized equipment.