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 usually needs 18-48 hours 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 need minimal instrumentation, but they only qualitatively indicate the presence or absence of enterococci, making comparison with quantitative contamination standards difficult. Here, we demonstrate semi-quantitative ‘thresholded’ LAMP via competition with pre-defined false targets to estimate the extent of enterococci contamination in water. The assay needs only one hour of incubation at a single temperature following which a simple visual examination of an oligonucleotide strand displacement (OSD) probe’s fluorescence allows order of magnitude scale estimation of enterococci contamination, correlating closely with the outcomes of both qPCR and plate cultures. Environmental water samples with low (undetectable by qPCR), medium (10s of DNA copies/mL), and high (1,000s of DNA copies/mL) enterococci contamination could be readily distinguished by thresholded LAMP-OSD, without interference from non-specific signals. The simplicity of implementing thresholded LAMP-OSD makes it well suited for point-of-need and low-cost water quality monitoring.