Inhibition challenges
@Zerina Rahic
Inhibition
PCR inhibition can affect eDNA samples from any environment (Jane et al., 2015), but the stagnant nature of ponds means they are particularly prone to inhibitor build-up. Ponds have high organic inputs due to dense vegetation, lack of water flow, and soil run-off, which encourages the build-up of algae, supports dense planktonic communities, and leads to high levels of natural turbidity. Turbid water with high suspended particulate matter not only clogs filters, but blocks extraction spin columns reducing DNA recovery. DNA extracts produced from turbid water often contain humic acid and tannin compounds, created through non-enzymatic decay of the organic material. These compounds can inactivate DNA polymerase and inhibit the PCR amplification process, reducing its efficiency or causing complete failure (Alaeddini, 2012; Albers et al., 2013; McKee et al., 2015).
PCR inhibition can cause false negatives, and thus it is imperative that eDNA practitioners and researchers test for it (Goldberg et al., 2016) using qPCR 123 32 Hydrobiologia (2019) 826:25–41 amplification of Internal Positive Controls (IPCs, see Table 1), such as Applied BiosystemsTM TaqMan Exogenous Internal Positive Control Reagents (Fig. 1, IPC3), or by spiking reactions with control DNA that will not be found in the sample (Doi et al., 2017).
Diluting eDNA extracts (Biggs et al., 2015; McKee et al., 2015) or reducing PCR template (Takahara et al., 2015) was previously recommended to overcome inhibition; however, we would not advise either approach. eDNA samples are characterised by low target DNA concentrations and dilution may ultimately reduce target DNA concentration below the limit of detection, causing false negatives despite diluting out inhibiting compounds (Buxton et al., 2017a). Use of droplet digital PCR (ddPCR, see Table 1) may overcome the aforementioned limitations for detection and quantification, particularly in turbid waters containing high concentrations of PCR inhibitors. In ponds, ddPCR outperformed qPCR, especially at very low eDNA concentrations (Doi et al., 2015a), and may be more accurate for abundance or biomass estimation due to lower variability (Nathan et al., 2014; Doi et al., 2015b)
Finally, in addition to running equipment, extraction and amplification blanks, and identification of inhibition using IPCs (Rees et al., 2014b; Goldberg et al., 2016), we recommend that quality control measures are taken to identify sample degradation and extraction efficiency (Fig. 1, IPC1 and 2). A known amount of non-target DNA can be introduced as IPC1 into ethanol precipitation sample kits before they are taken into the field, or non-target DNA can be introduced into a preservative solution for filtered samples. IPC2 can be added before or during the first step of DNA extraction. In similar fashion to IPC3,this sequence would be targeted during qPCR and failure to amplify, or amplification after more cycles than expected, would indicate sample degradation or low extraction efficiency. This will help improve confidence in negative results.
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