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In this study, we optimized and validated a chip-based dPCR EvaGreen assay with commonly used 16S rRNA gene primer pairs and compared its performance to quantitative real-time PCR (qPCR). We compared measurements of low amounts of template DNA using a newly designed synthetic DNA standard to assess precision, accuracy, and sensitivity. Optimization approaches were tested to minimize partitions with intermediate fluorescence levels between true positive and true negative partitions (so-called \u201crain\u201d) for dPCR. Both dPCR and qPCR demonstrated similar quantification performance, with variability in accuracy increasing for samples containing fewer than 30 copies \u03bcl\u22121 template concentrations. Both tested 16S rRNA gene primer sets amplified non-target template contaminants within both qPCR and dPCR mixtures, which could not be eliminated by ultraviolet light or DNAse treatment and negatively affected the apparent sensitivity of both PCR assays. Digital PCR was less susceptible to common PCR inhibitors, such as ethanol and humic acids, but was more susceptible to tannic acid inhibition than qPCR. These findings demonstrate the suitability of dPCR for 16S rRNA gene quantification of low biomass environmental samples.<\/jats:p>","DOI":"10.1093\/ismeco\/ycaf115","type":"journal-article","created":{"date-parts":[[2025,7,9]],"date-time":"2025-07-09T14:33:19Z","timestamp":1752071599000},"source":"Crossref","is-referenced-by-count":7,"title":["Validating digital polymerase chain reaction for 16S rRNA gene amplification from low biomass environmental samples"],"prefix":"10.1093","volume":"5","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-6000-8886","authenticated-orcid":false,"given":"Veronika V","family":"Koziaeva","sequence":"first","affiliation":[{"name":"Department of Biology, University of Waterloo , Waterloo, ON N2L 3G1 ,","place":["Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4141-3819","authenticated-orcid":false,"given":"Katja","family":"Engel","sequence":"additional","affiliation":[{"name":"Department of Biology, 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