Development of A Rapid, Low-Cost Portable Detection Assay for Enterococci in Wastewater and Environmental Waters

Waterborne diseases are known as a leading cause of illness and death in both developing and developed countries. Several pathogens can be present in contaminated water, particularly waters containing faecal material; however, routine monitoring of all pathogens is not currently possible. Enterococcus faecalis, which is present in the microflora of human and animals has been used as a faecal indicator in water due to its abundance in surface water and soil. Accurate and fast detection methods are critical for the effective monitoring of E. faecalis in the environment. Although conventional and current molecular detection techniques provide sufficient sensitivity, specificity and throughput, their use is hampered by the long waiting period (1-6 days) to obtain results, the need for expensive laboratory equipment, skilled personnel, and cold-chain storage. Therefore, this study aimed to develop a detection system for E. faecalis that would be simple, rapid, and low-cost, using an isothermal DNA amplification assay called recombinase polymerase amplification (RPA), integrated with a lateral flow assay (LFA). The assay was found to be 100% selective for E. faecalis and capable of detecting rates as low as 2.8 × 10³ cells per 100 mL from water and wastewater, and 2.8 × 10⁴ cells per 100 mL from saline water. The assay was completed in approximately 30 min using one constant temperature (38 °C). In addition, this study demonstrated the quantitation of E. faecalis using a lateral flow strip reader for the first time, enhancing the potential use of RPA assay for the enumeration of E. faecalis in wastewater and heavily contaminated environmental waters, surface water, and wastewater. However, the sensitivity of the RPA-LFA assay for the detection of E. faecalis in tap water, saline water and in wastewater was 10-1000 times lower than that of the Enterolert-E test, depending on the water quality. Nevertheless, with further improvements, this low-cost RPA-LFA may be suitable to be used at the point-of-need (PON) if conjugated with a rapid field-deployable DNA extraction method.