Formatted Title
Exploring Digital Droplet PCR as a Third-Generation Quantification Method for Rapid and Sensitive Biomonitoring of Reductive Dechlorination Biomarker
Background/Objectives
Molecular biological tools (MBTs) are commonly employed for quantifying biomarker genes in the characterization and biomonitoring of contaminated sites, the development of new biotechnologies and processes, and studies related to the cultivation of dechlorinating consortia. Over the past two decades, quantitative real-time PCR (qPCR) has been the preferred MBT for quantifying Dehalococcoides mccartyi (Dhc) and reductive dehalogenase genes, which are crucial biomarkers for the reductive dechlorination (RD) process. Indeed, within the context of bioremediation projects targeting chlorinated solvent-contaminated sites, quantitative data within specific ranges provide an initial assessment of the bioremediation potential of the site. Based on our extensive experience analyzing a large number of contaminated samples (≥ 3 thousand in the last ten years) we have observed that although qPCR is a valid MBT for quantifying dechlorinating biomarkers, its sensitivity, accuracy, and precision decrease at low abundances (i.e., ≤ 1E+04 or 1E+03 gene copies/L). Therefore, a detection limit of 1E+03 gene copies/L is crucial, and values below this threshold should be interpreted with caution. This is particularly important for contaminated samples with low Dhc abundance, as it can lead to erroneous evaluations of the bioremediation potential of the site. In this study, we present novel assays for rapid and accurate ddPCR quantification to target 16S rRNA Dhc and reductive dehalogenase genes.
Approach/Activities
We developed the ddPCR protocol for the 16S rRNA Dhc, reductive dehalogenase genes (i.e. tceA, bvcA, vcrA), and some other functional genes used as biomarkers for the oxidative dechlorination (i.e., etnE, etnC). The ddPCR protocol was rigorously tested on various samples, including dechlorinating consortia, environmental samples, and long amplicons commonly employed as qPCR standards. To assess the performance of ddPCR, we compared the quantitative data obtained using this technique with the results obtained from qPCR within the same concentration range (ranging from 1E+10 to 1 gene copy/reaction).
Results/Lessons Learned
ddPCR demonstrated exceptional precision and sensitivity. Notably, it outperformed qPCR in reliability and reproducibility when quantifying gene copies below 1E+06 copies/reaction, extending down to 1 gene copy/reaction. In contrast, within the range of 1E+10 to 1E+06 gene copies/reaction, ddPCR reached its upper quantification limit due to excessive target concentrations, requiring sample dilution. qPCR results below 1E+03 or 1E+02 gene copies/reaction were unreliable. This study compared ddPCR and qPCR for quantifying biomarkers in reductive and oxidative dechlorination. The results revealed that ddPCR's dynamic range spans from 1E+06 gene copies/reaction to 1 gene copy/reaction, indicating superior sensitivity. ddPCR offers advantages such as heightened sensitivity, enhanced precision for low target concentrations, and improved reliability across a wide concentration range, especially at low target abundances. These results underscore the potential of ddPCR to improve the accuracy of biomonitoring in chlorinated solvent-contaminated sites, aiding in more informed decision-making for effective bioremediation strategies.