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

Abstract

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.

Keywords: Enterococcus faecalis; lateral flow assay; recombinase polymerase amplification; wastewater; water contamination.

Figure 1

Screening of primers: Lanes 1 and 2—Ent rpoB F1/R1, Lanes 3 and 4—Ent rpoA F1/R1, Lane 5 and 6—Enttuf F1/R1, Lane 7 and 8—Enttuf F2/R2. Lane M—100 bp molecular ladder. (Note: primer concentration is −10 µM, template—2 µL, assay temperature and time—38 °C for 20 min).

Figure 2

Lateral flow assay: RPA-LFA using the Ent rpoB F1/R1 primer set with positive template E. faecalis DNA (T), and non-template control (NTC).

Figure 3

Specificity validation of Ent F1/R1 primer set using RPA-AGE. Lane M: 100 bp molecular marker, Lanes 1 to 10: E. faecalis, E. faecium, Staphylococcus aureus, Streptococcus pneumoniae, Bacillus cereus, Pseudomonas aeruginosa, E. coli O157:H7, Salmonella typhimurium, Shigella dysenteriae, E. coli K-12.

Figure 4

Sensitivity of RPA assay for detection of E. faecalis using (A) tap water; (B) Koo Wee Rup wastewater; (C) Lang Lang wastewater; (D) saline. Well M: molecular marker; Wells 1 to 7: 2.8 × 10⁶ CFU/100 mL, 2.8 × 10⁵ CFU/100 mL, 2.8 × 10⁴ CFU/100 mL, 2.8 × 10³ CFU/100 mL, 2.8 × 10² CFU/100 mL, 2.8 × 10¹ CFU/100 mL, 2.8 CFU/100 mL.

Figure 5

Confirmation of the presence of E. faecalis in Lang Lang wastewater. Lanes M and 11: molecular marker, Lanes 1–8: RPA-AGE of Lang Lang wastewater neat, 10⁻¹, 10⁻², 10⁻³, 10⁻⁴, 10⁻⁵, 10⁻⁶, 10⁻⁷. Lanes 12–19: PCR of Lang Lang wastewater neat, 10⁻¹, 10⁻², 10⁻³, 10⁻⁴, 10⁻⁵, 10⁻⁶, 10⁻⁷.

Figure 6

(A) Comparison of the RPA-LFA visual outputs and the values recorded by the GT810. (B) Standard curve plot of bacterial cell numbers v GT810 readings; (•) represents the value produced by the LF reads corresponding to the CFU count of bacteria per 100 mL of growth medium.