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Updated: Sep 19, 2025
A Duplex Digital PCR Assay for Simultaneous Quantification of the Enterococcus spp. and the Human Fecal-associated HF183 Marker in Waters
Published on: March 9, 2016
A streamlined qPCR method for characterization of Enterococcus spp. levels in ambient surface water samples
Mano Sivaganesan1, Stephanie A Dean1, Jessica R Willis1
1US. Environmental Protection Agency, Office of Research and Development, Center for Environmental Measurement and Modeling, 26 West Martin Luther King Drive, Cincinnati, OH 45268, USA.
A new streamlined quantitative polymerase chain reaction (qPCR) protocol for Enterococcus testing offers faster, safer, and more reproducible recreational water quality assessments. This method improves upon traditional culture-based techniques, providing same-day results and reducing errors.
Area of Science:
- Environmental microbiology
- Molecular biology
- Water quality assessment
Background:
- Traditional culture-based methods for Enterococcus spp. detection in recreational waters require over 18 hours for results.
- Quantitative polymerase chain reaction (qPCR) offers same-day results but existing protocols can be improved.
- Advances in qPCR technology present an opportunity to update current Enterococcus testing methodologies.
Purpose of the Study:
- To introduce a streamlined Enterococcus qPCR protocol that simplifies calculations and reduces testing time.
- To incorporate a certified control material and an inactivated whole cell DNA standard (WCDS) for enhanced accuracy and safety.
- To validate the new protocol against existing methods and assess alternative controls for water sample matrix interference.
Main Methods:
- Comparison of a new streamlined Enterococcus qPCR protocol with the 2015 EPA protocol using 60 marine, estuarine, and freshwater samples.
- Investigation of alternative sources for sample processing control baseline measurements.
- Evaluation of the performance, homogeneity, and stability of an inactivated E. faecalis whole cell DNA standard (WCDS).
Main Results:
- Strong correlation (R² = 0.980) and 100% agreement in amplification and control tests between protocols.
- 72.7% of samples showed reduced error with the streamlined protocol, confirmed by Bayesian analysis.
- Inactivated WCDS demonstrated statistically equivalent performance, reproducibility, and stability compared to viable controls.
Conclusions:
- The streamlined Enterococcus qPCR protocol is faster, easier, safer, and more reproducible than existing methods.
- Alternative control sources effectively adjusted for water sample matrix interference without impacting results.
- The inactivated E. faecalis whole cell DNA standard (WCDS) is a reliable and stable control material for Enterococcus qPCR testing.
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