Loop-Mediated Isothermal DNA Amplification for the On-Site Detection of Human Pathogenic Legionella from Water Samples
Legionella are bacteria that live in water. The rapid detection of these microorganisms, some of which are pathogenic to humans, can be achieved by means of loop-mediated isothermal amplification (LAMP). In this process, a species-specific part of the genetic material of the pathogens is amplified. For this purpose, methods that allow the pathogens to be quantified were developed at Leibniz IPHT to potentially be used for simple and inexpensive on-site detection.
Legionella are widespread bacteria found in nature in both fresh water and drinking water systems, some of which are pathogenic to humans. The species Legionella pneumophila has a special significance as a causative agent of Legionnaires’ disease. A contamination of domestic drinking water installations requires immediate action to prevent an aerosol-based infection of the lungs. The current standard for the detection of Legionella is a time-consuming cultivation process, which prevents quick action in the case of contamination. Therefore, the aim of our current research was to develop rapid, on-site detection methods for these human pathogens. For this purpose, a colorimetric method was combined with isothermal LAMP-based DNA amplification (Blue-LAMP). Detection is based on the optical signal of the dye hydroxynaphthol blue (HNB). LAMP produces a comparatively strong formation of amplificates and pyrophosphate, the latter of which forms a complex with magnesium ions. The resulting decrease in magnesium concentration leads to a color change in the metal indicator HNB from violet to blue. This color signal allows the detection of DNA amplification with the naked eye. In addition, LAMP is more robust, faster, and requires less complex equipment than the comparable standard polymerase chain reaction (PCR) method. Thus, LAMP can be performed in a miniaturized format (here on a stationary chip) and without complex technical effort.
In a further approach, detection is implemented in the form of a droplet-based digital assay (droplet digital LAMP – ddLAMP), which, with the help of Poisson statistics, allows a quantitative assessment of the initial DNA concentration and thus the pathogen quantity. The Blue-LAMP was optimized with regard to reaction additives and other factors, such as the concentrations of MgSO4 and HNB, the gene region used (thus different LAMP primer systems of at least four primers per target sequence), and the incubation temperature. The reaction was then tested regarding the detection limit. With an incubation time of about 45 minutes at 65°C, a DNA concentration of about 1 pg/µl was able to be reproducibly detected which corresponds to about 270 molecules.
In the picture:
Isothermal amplification of the genetic material of L. pneumophila using ddLAMP allows an exact quantification of the pathogen DNA by evaluating the fluorescence signal in the individual droplets (left). By miniaturization on the stationary chip (total droplet size less than 15 µl) a reproducible detection of the pathogens can be achieved in a batch of only 3 µl (right).
The specificity and selectivity of the detection method was investigated for L. pneumophila in comparison to other relevant Legionella species and drinking water microorganisms in a variety of tests. Overall, the LAMP-based approaches show a high specificity and sensitivity. Due to simple, laboratory-independent feasibility and short reaction time in combination with high efficiency, the newly developed Blue-LAMP method can potentially be used as a rapid test for monitoring water quality.
In the image above:
Fluorescence micrograph of a reaction batch of loop-mediated isothermal DNA amplification (ddLAMP): the orange dye shows the droplets with successful amplification.