Challenges Using Droplet Digital PCR for Environmental Samples

Abstract

Droplet digital polymerase chain reaction (ddPCR) is a method used to detect and quantify nucleic acids even when present in exceptionally low numbers. While it has proven to be valuable for clinical studies, it has failed to be widely adopted for environmental studies but despite some limitations, ddPCR may represent a better option than classical qPCR for environmental samples. Due to the complexity of the chemical and biological composition of environmental samples, protocols tailored to clinical studies are not appropriate, and results are difficult to interpret. We used environmental DNA samples originating from field studies to determine a protocol for environmental samples. Samples included field soils which had been inoculated with the soil fungus Rhizophagus irregularis (environmental positive control), field soils that had not been inoculated and the targeted fungus was not naturally present (environmental negative control), and root samples from both field categories. To control for the effect of soil inhibitors, we also included DNA samples of an organismal control extracted from pure fungal spores (organismal positive control). Finally, we included a no-template control consisting only of the PCR reaction reagents and nuclease free water instead of template DNA. Using original data, we examined which factors contribute to poor resolution in root and soil samples and propose best practices to ensure accuracy and repeatability. Furthermore, we evaluated manual and automatic threshold determination methods and we propose a novel protocol based on multiple controls that is more appropriate for environmental samples.

Assessing the performance and efficiency of environmental DNA/RNA capture methodologies under controlled experimental conditions

“…The stronger ddPCR signal reported from the low concentration cell treatments can be attributed to better discrete cloud clustering and therefore higher amplitude level readout in diluted samples (Kokkoris et al, 2021). The negative effect of high target concentration was especially evident in RNA dataset; however, the pattern of membrane performance remained consistent across treatments, suggesting no or limiting impact of that factor on efficiency assessment results.…”

“…This is consistent with results from previous studies, where the influence of dissolved substances in the water column on DNA detection rates has been reported, and explained by PCR inhibition effects (Albers et al, 2013; Hunter et al, 2019) or nucleic acids binding to suspended particles (Stoeckle, Beggel, et al, 2017). The stronger ddPCR signal reported from the low concentration cell treatments can be attributed to better discrete cloud clustering and therefore higher amplitude level readout in diluted samples (Kokkoris et al, 2021). The negative effect of high target concentration was especially evident in RNA dataset; however, the pattern of membrane performance remained consistent across treatments, suggesting no or limiting impact of that factor on efficiency assessment results.…”

Diagnostic, Prognostic, and Therapeutic Value of Droplet Digital PCR (ddPCR) in COVID-19 Patients: A Systematic Review

“…Multiple parameters need to be adjusted, as they could impact the sensitivity of ddPCR. These include the amount of input genetic material, the concentration of the primers used in the reaction, the cycling conditions (e.g., duration of denaturation, annealing, and extension; number of cycles; temperature during annealing and extension), also determining the fluorescence threshold and LOD [ 62 ]. Of interest, a study showed that undigested fragments could fail packaging into droplets, compromising the performance of the assay [ 63 ].…”

“…Even if less likely than RT-qPCR, ddPCR detection ability could also be impacted by inhibitors or non-target genetic material. Thus, optimization of the ddPCR assay and strict quality control measures are needed to achieve accurate and reproducible results [ 58 , 62 ]. Improved artificial intelligence algorithms could also help towards this direction [ 64 ].…”

Molecular Methods for Pathogenic Bacteria Detection and Recent Advances in Wastewater Analysis

“…In comparison to qPCR, ddPCR shows better performance in detecting low concentrations of target genes in environmental samples. Moreover, it has the potential to reduce the effect of qPCR inhibitors, although its application to complex environmental samples needs further optimization [111]. However, qPCR is more reliable in detecting higher concentrations (2 × 10 5 or 2 × 10 4 gene copies/PCR), since ddPCR displays higher variability and less precision in these concentration ranges [112].…”