Enzymatic inhibition
DNA extracted from environmental samples is known to potentially include co-extracted enzymatic inhibitors, which negatively affect the polymerase in PCR and can lead to underestimation of original target DNA copies as well as false negatives . Different strategies have been proposed to mitigate the adverse effects of inhibitors, including the dilution of template DNA used and/or the addition of inhibition-reducing agents such as Bovine Serum Albumin (BSA). Thus, since a dilution-like effect of template DNA is already expected when carrying out qPCR reaction at nano-liter scales (as is the case for droplet digital PCR (ddPCR) as well as the microfluidic-enabled HT-qPCR system used in the present study), the inhibitor-resistant effects of adding BSA to the intended HT-qPCR protocol was tested against two well-known inhibiting substances (i.e. humic acid and EDTA) with the aid of Internal Amplification Control (IAC) assays . For this step, 48.48 IFC configurations were used (using protocol QR 100-9791Rev3), whereby the qPCR amplification was assessed with or without the addition of BSA (at 1 µg/µl concentration). To test for potential effects on different concentrations of target DNA, three distinct IACs were tested at 100000, 10000 and 1000 copies/µl, respectively, whereas to test for the effect of BSA against different concentrations of inhibiting substances, five concentrations of humic acid (2500, 1250, 625, 312.5, 156.25 µM) and five of EDTA (40, 20, 10, 5, 2.5 ng/µl) were tested. All reactions were carried out in triplicate, and concentration ranges of inhibiting substances and evidence of inhibition in the form of delays or absence of amplification in spiked samples (n = 30) vs non-spiked samples (n = 30) were chosen based on previous studies , with HT-qPCR conducted using 48.48 IFCs.