Fieldable isothermal nucleic acid test for rapid semi-quantitative visual readout of Enterococci in recreational waters

Enterococci are fecal indicator bacteria whose presence in water suggests the potential for gastrointestinal pathogens. Culture-based detection is slow and may require expensive apparatus and proprietary media. Molecular tests based on qPCR can be technically difficult and require complex devices. Loop-mediated isothermal amplification (LAMP) tests that need minimal instrumentation have been described but they only qualitatively indicate presence or absence of Enterococci, which is difficult to compare with quantitative contamination standards for assessing water quality. Here, we demonstrate semi-quantitative thresholded LAMP that uses internal competition with pre-defined false targets and an oligonucleotide strand displacement (OSD) probe to measure the degree of Enterococcus contamination in water. The assay needs only one hour of incubation at a single temperature following which a simple visual examination of endpoint OSD fluorescence allows order of magnitude scale estimation of Enterococcus amounts. Environmental water samples with low, medium, and high Enterococcus contamination could be readily distinguished by thresholded LAMP-OSD without interference from non-specific signals. LAMP-OSD performance correlated with the outcomes of both qPCR and plate cultures of the samples. The simplicity of implementing thresholded LAMP-OSD makes it well suited for point-of-need and low-cost water quality monitoring.