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Validating digital polymerase chain reaction for 16S rRNA gene amplification from low biomass environmental samples
Veronika V Koziaeva1, Katja Engel1, Josh D Neufeld1
1Department of Biology, University of Waterloo, Waterloo, ON N2L 3G1, Canada.
ISME Communications
|August 13, 2025
Summary
Digital polymerase chain reaction (dPCR) offers absolute DNA quantification. This study validated a chip-based dPCR EvaGreen assay for 16S rRNA gene quantification, showing its suitability for low biomass environmental samples.
Area of Science:
- Molecular Biology
- Microbiology
Background:
- Digital polymerase chain reaction (dPCR) provides absolute DNA quantification.
- 16S rRNA gene sequencing is crucial for microbial community analysis.
Purpose of the Study:
- To optimize and validate a chip-based dPCR EvaGreen assay for 16S rRNA gene quantification.
- To compare dPCR performance against quantitative real-time PCR (qPCR) for low-template DNA concentrations.
Main Methods:
- Optimization of a chip-based dPCR EvaGreen assay using 16S rRNA gene primer pairs.
- Comparison of dPCR and qPCR using a synthetic DNA standard for accuracy, precision, and sensitivity assessment.
- Evaluation of PCR inhibitor effects on dPCR and qPCR.
Main Results:
- Both dPCR and qPCR showed similar quantification performance, with accuracy decreasing below 30 copies μl-1.
- Non-target template amplification affected sensitivity in both assays.
- dPCR exhibited differential susceptibility to PCR inhibitors compared to qPCR.
Conclusions:
- Chip-based dPCR is suitable for quantifying 16S rRNA genes in low biomass environmental samples.
- Optimization is necessary to minimize 'rain' and improve assay reliability.
- Understanding inhibitor effects is crucial for accurate dPCR and qPCR applications.
