J. Li, X. Lin, X. Huang, Y. Zhu, K. Urmann, M.R. Hoffmann
California Institute of Technology,
United States
Keywords: pathogen detection, typhoid, digital LAMP, asymmetric membrane, nanofluidics
Summary:
A simple and cost-effective membrane based on digital loop-mediated isothermal amplification (mLAMP) method was developed to simultaneously concentrate and detect Salmonella Typhi in large-volume of drinking water samples (Figure 1). Compared to traditional real-time polymerase chain reaction (qPCR) method, mLAMP shows higher tolerance against inhibitors (e.g., heavy metals, organic matters) and provides absolute quantification results without the need of standard curve calibration. Briefly, mLAMP holds great potential for microbial water quality analysis in resource-limited settings. The bilayer asymmetric membrane, as shown in Figure 2, was prepared by combining two commercial polycarbonate (PC) membranes of different pore sizes (top: 25 μm and bottom: 600 nm) through simple heating at 170°C. Drinking water samples spiked with different concentrations of Salmonella Typhi were used as model water and tested with the mLAMP method. For each test, 100 mL water sample were filtered through the bilayer membrane as shown in Figure 3. Bacteria were enriched and randomly partitioned into the top 25μm micropores, while smaller particles were washed away through the bottom 600-nm nanopores. The filtered asymmetric membrane was then loaded with the LAMP reagents, followed by heat incubation at 65°C for 60 min for nucleic acid amplification. The endpoint fluorescence images of the asymmetric membrane were taken using a fluorescence microscope. The concentrations of Salmonella Typhi in original samples were calculated by the number of positive pores (pores exhibit increased fluorescence, as shown in Figure 4) and the total number of pores using Poisson distribution. For comparison, the same samples were also filtered and concentrated through a single layer 0.2 μm PC membranes. The attenuated cells were mechanically lysed using bead-beating method. Subsequently, cell DNA was extracted and then analyzed using qPCR following the protocol described in Nga et al. (2010). It took around 10 min for the asymmetric membrane to filter up to 100 mL water sample and the recovery rate of Salmonella Typhi cells was around 60%. The detection limit of mLAMP was as low as 0.6 copies/mL, which was 10-fold lower than qPCR (Figure 5). Additionally, as the DNA extraction step was not necessary in mLAMP, the sample-to-answer time was shortened from 4-5 h to 1-2 h. Our preliminary study provides a novel analytical method for Salmonella Typhi quantification in large-volume of water samples. This development has a promising prospect of being applied for microbial water quality analysis in low-resource settings due to its portability, cost-effectiveness and user-friendliness.