Quantitative Estimation of Low-Abundance Targets in Engineered Systems and Environmental Samples: Comparative Study Between Droplet Digital PCR and Real-Time PCR

Real-Time PCR (qPCR) is an extensively used biomolecular tool for the detection and quantification of nucleic acids for a variety of applications, spanning from clinical to environmental settings. However, qPCR relies on an external calibration curve and can be susceptible to inhibition caused by pollutants that are commonly found in environmental samples. More recently, droplet digital PCR (ddPCR) was proven to be the method of choice for detection and quantification when a target is present at a low abundance. While it has been extensively utilized in clinical studies, only a small amount of data is available for complex samples, which are often characterized by a low target concentration and high abundances of non-target and PCR inhibitors. In this study, ddPCR and qPCR assays were performed on the same DNA serial dilutions with both Eva/SYBR Green and TaqMan chemistry. The comparative analysis was conducted on seven different samples taken from environmental and engineered settings. Ammonia-oxidizing bacteria (AOB) were chosen as the target, as they are ubiquitous and widespread and responsible for a fundamental environmental process in the global biogeochemical nitrogen cycle and in engineered settings such as wastewater treatment plants (WWPTs). ddPCR produced precise, reproducible, and statistically significant results in all samples, also showing an increased sensitivity to detecting AOB in complex samples characterized by low levels of the target and low target/non-target ratios.