Development of A Rapid, Low-Cost Portable Detection Assay for Enterococci in Wastewater and Environmental Waters
Abstract
:1. Introduction
2. Material and Methods
2.1. Samples
2.2. Bacterial Isolates
2.3. Standard Plate Count for Enterococcus faecalis
2.4. Enterolert-E Test
2.5. Total DNA Extraction from Selected Bacteria
2.6. Designing and Screening of RPA Primers
2.7. Recombinase Polymerase Amplification-Agarose Gel Electrophoresis (RPA-AGE)
2.8. Recombinase Polymerase Amplification-Lateral Flow Assay (RPA-LFA)
2.9. Selectivity and Sensitivity of Recombinase Polymerase Amplification (RPA)
2.10. Quantification of the Recombinase Polymerase Amplification-Lateral Flow Assay (RPA-LFA) Using the Benchtop Reader
3. Results and Discussion
3.1. DNA Extraction and Selection of the Primers
3.2. Selectivity Assessment of the RPA Using Selected Primer Set
3.3. Sensitivity of the Recombinase Polymerase Assay and Impact of Environmental Water Characteristics
3.4. Potential for RPA-LFA as a Quantitative Measurement System for the Enumeration of E. faecalis
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Primer Name | Targeted Gene | Oligonucleotide Sequence (5′ to 3′) | Direction |
---|---|---|---|
Enttuf F1 | tuf | TGACGATAAAGAAGAGAGCGGAGACACGAATC | Forward |
Enttuf R1 | tuf | CCCCATCTTTTTCATTTGGAGCGATAGTTTTT | Reverse |
Enttuf F2 | tuf | GACGATAAAGAAGAGAGCGGAGACACGAATCC | Forward |
Enttuf R2 | tuf | GCCCCATCTTTTTCATTTGGAGCGATAGTTTT | Reverse |
Ent rpoA F1 | rpoA | GCAGTGAAAACCGAAGCAAGCGCCATTCAAAT | Forward |
Ent rpoA R1 | rpoA | TAAGGCATCAAATTCTTCCTCCAATAAAATAT | Reverse |
Ent rpoB F1 F1 | rpoB | GGACCCGCTACCGTGACTGCCGGCGATATTATCG | Forward |
Ent rpoB R1 R1 | rpoB | Biotin-GAATCAACTGGAAGTACACCGATTGGCATATC | Reverse |
Ent rpoB P1 | 5′-FAM -GATGTTGAGATCCTAAATAAAGATTTAGTTAT/dSpacer/TGTAGTGTTGCTGAAGGAG-C3 spacer-3′ | Probe |
Sample Dilution (from A Culture Containing 2.8 × 108 Organisms/100 mL) Calculated Using Selective Isolation Plating. Actual Numbers are Shown in Brackets | Estimated Numbers in Spiked Tap Water | Estimated Numbers in Spiked Saline | Estimated Numbers in Spiked Koo Wee Rup Wastewater | Estimated Numbers in Non-Spiked Lang Lang Wastewater |
---|---|---|---|---|
Control (0/100 mL) | 0 | 0 | 0 | 0 |
10−6 (2.8 × 100) | 0 | 0 | 1222.0–1986.3 | 0–1 |
10−5 (2.8 × 101) | 120–175 | 0 | 1630.4–2419.6 | 0.7–3.1 |
10−4 (2.8 × 102) | 1203–1750 | 20–30.4 | >2419.6 to infinite | 55.0–77.1 |
10−3 (2.8 × 103) | >2419.6 to infinite | 143.7–181.9 | >2419.6 to infinite | 126.1–187.2 |
10−2 (2.8 × 104) | >2419.6 to infinite | >2419.6 to infinite | >2419.6 to infinite | 120.8–179.3 |
10−1 (2.8 × 105) | >2419.6 to infinite | >2419.6 to infinite | >2419.6 to infinite | 126.1–187.2 |
100 (2.8 × 106) | >2419.6 to infinite | >2419.6 to infinite | >2419.6 to infinite | 206.6–325.5 |
Technique | Description | Advantages | Disadvantages | Website |
---|---|---|---|---|
Selective Plate Isolation | Use of a dilution series of sample plated onto selective isolation media enabling the counting of colonies present (colony-forming units). | Simple detection system. Can be accurate. | Time consuming—24–48 h. Requires experienced laboratory technician and laboratory. Expensive in terms of consumables. May underestimate the number of enterococci in samples with high turbidity. | https://www.merckmillipore.com/AU/en/products/industrial-microbiology/culture-media/, (accessed on 20 November 2019) |
Membrane Filtration | Use of membranes (pore size 0.22 −0.45 µm) to filter water after following by selective isolation plating (colony-forming units). | Efficient system. Can be accurate. | Time consuming—24–48 h. Requires experienced laboratory technician and laboratory. Expensive in terms of consumables. May underestimate the number of enterococci in samples with high turbidity. | www.oxoid.com/UK/blue/prod_detail/, (accessed on 20 November 2019) |
IDEXX Enterolert-E | Based on the use of a defined substrate technology nutrient indicator to detect enterococci. This nutrient indicator fluoresces when metabolised by enterococci. (most probable number of cells). | No media preparation. Sensitive to 1 enterococcus per 100 mL. Less subjective interpretation. 50% fewer false positives and 95% fewer false negatives than the standard membrane filtration (MF) method. | Time consuming—24–48 h. Requires experienced laboratory technician and laboratory. Expensive in terms of consumables. Overestimation/underestimation of Enterococci is possible. | https://www.idexx.com.au/en-au/water/water-products-services/enterolert/, (accessed on 24 November 2019) |
Polymerase Chain Reaction | Based on the use of 16S rDNA real-time PCR (gene copies). | Relatively fast (3–5 h), specific and sensitive. Multiplex PCR is possible. Quantitative detection (qPCR) of target pathogen is rapid (number of gene copies). | Well-equipped laboratory required. Requires primer optimisation and DNA extraction. Requirement of skilled personnel. | https://www.genesig.com/, (accessed on 4 December 2019) |
Recombinase Polymerase Amplification | Versatile isothermal DNA/RNA amplification by TwistDx DNA polymerase. Based on the use of rpoB (RNA polymerase subunit B) specific gene target. | Rapid, less than 1 h.Carried out at 37–42 °C Requires very limited equipment and training. | Requires primer optimisation and DNA extraction. | http://www.twistdx.co.uk, (accessed on 21 September 2020) |
Parameters | Sensitivity (CFU/100 mL) | Time Required | PON Applicability | Approximate Cost USD/Reaction | Temperature Required | References | |
---|---|---|---|---|---|---|---|
Assay | |||||||
PCR | 104–105 | 120–150 min | NP | 5 | Thermal cycling (95 °C, 55 °C, 72 °C) | [39,40] | |
RPA-EF | 2.8 × 103–2.8 × 103 | 80 min | NP | 5.5 | 38 °C | This study | |
RPA-LFA | 2.8 × 103 | 25–30 min | P | 6 | 38 °C | This study |
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Batra, A.R.; Cottam, D.; Lepesteur, M.; Dexter, C.; Zuccala, K.; Martino, C.; Khudur, L.; Daniel, V.; Ball, A.S.; Soni, S.K. Development of A Rapid, Low-Cost Portable Detection Assay for Enterococci in Wastewater and Environmental Waters. Microorganisms 2023, 11, 381. https://doi.org/10.3390/microorganisms11020381
Batra AR, Cottam D, Lepesteur M, Dexter C, Zuccala K, Martino C, Khudur L, Daniel V, Ball AS, Soni SK. Development of A Rapid, Low-Cost Portable Detection Assay for Enterococci in Wastewater and Environmental Waters. Microorganisms. 2023; 11(2):381. https://doi.org/10.3390/microorganisms11020381
Chicago/Turabian StyleBatra, Alka Rani, Darren Cottam, Muriel Lepesteur, Carina Dexter, Kelly Zuccala, Caroline Martino, Leadin Khudur, Vivek Daniel, Andrew S. Ball, and Sarvesh Kumar Soni. 2023. "Development of A Rapid, Low-Cost Portable Detection Assay for Enterococci in Wastewater and Environmental Waters" Microorganisms 11, no. 2: 381. https://doi.org/10.3390/microorganisms11020381
APA StyleBatra, A. R., Cottam, D., Lepesteur, M., Dexter, C., Zuccala, K., Martino, C., Khudur, L., Daniel, V., Ball, A. S., & Soni, S. K. (2023). Development of A Rapid, Low-Cost Portable Detection Assay for Enterococci in Wastewater and Environmental Waters. Microorganisms, 11(2), 381. https://doi.org/10.3390/microorganisms11020381