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.