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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
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Absolute Quantification of Enterococcal 23S rRNA Gene Using Digital PCR.
Dan Wang1, Kevan M Yamahara1, Yiping Cao2
1Environmental and Water Studies, Department of Civil and Environmental Engineering, Stanford University , Stanford, California 94305, United States.
Environmental Science & Technology
|February 24, 2016
Summary
Chip-based digital PCR (dPCR) accurately quantifies enterococci in water, offering a precise alternative to quantitative PCR (qPCR) for water quality monitoring. This method overcomes challenges with redundant genes and shows improved precision at relevant concentrations.
Area of Science:
- Environmental Microbiology
- Molecular Biology
- Water Quality Analysis
Background:
- Enterococci are key fecal indicators for water quality monitoring, recommended by the USEPA.
- Accurate quantification of enterococci is crucial for assessing recreational water safety.
- Digital PCR (dPCR) offers a potential advancement in DNA quantification methods.
Purpose of the Study:
- To evaluate the efficacy of chip-based digital PCR (dPCR) for quantifying enterococci in water samples.
- To compare dPCR performance against quantitative PCR (qPCR) and cell counts.
- To assess the impact of common inhibitors on dPCR accuracy.
Main Methods:
- Utilized chip-based digital PCR (dPCR) to enumerate enterococci by targeting the 23s rRNA gene.
- Extracted genomic DNA (gDNA) using commercial kits.
- Compared dPCR results with quantitative PCR (qPCR) and direct cell counts using BioBall standards and environmental water samples.
- Investigated the effects of humic acid and calcium on dPCR and qPCR inhibition.
Main Results:
- dPCR accurately quantified enterococci, with results closely matching fluorometer measurements (R² = 0.99).
- dPCR quantification correlated strongly with qPCR (R² = 0.96) and cell counts.
- dPCR demonstrated higher precision (narrower 95% confidence intervals) than qPCR at environmentally relevant concentrations.
- Calcium showed less inhibition in dPCR compared to qPCR; inhibition in dPCR was partially mitigated by increasing thermal cycles.
Conclusions:
- Chip-based digital PCR (dPCR) is a viable and accurate method for enumerating enterococci in ambient water.
- dPCR provides a precise alternative to qPCR for water quality monitoring, especially for redundant gene targets.
- Understanding inhibitor effects is crucial for optimizing dPCR application in environmental monitoring.
