Reagents - PCR inhibition
@Zerina Rahic
PCR inhibition
Some studies reported significant inhibitory effects on PCR amplification leading to false‐negative results. Such inhibitory effects can also induce uncertain quantification of eDNA concentration, which would hinder attempts to estimate the abundance and biomass of target organisms based on the amount of DNA. Thus, avoiding inhibition in PCR amplification is a key to realizing robust estimation of species distribution by real‐time PCR‐based eDNA assays.
Inhibition of PCR amplification occurs due to the inhibition of DNA polymerase activity, which can be caused either directly or indirectly by a variety of substances. One of them are:
- Heat
- Humic, fulvic and tannic acid
The use of inhibitor‐resistant PCR reagents is advantageous because it is the easiest way to avoid PCR inhibition without any additional procedures in sample preparation.
Six PCR reagents were tested in this study:
- TaqMan Gene Expression Master Mix (GMM)
- TaqMan Environmental Master Mix 2.0 (EMM)
- TaqMan Fast Advanced Master Mix (FMM)
- TaqPath qPCR Master Mix (TMM)
- CG (TMM) from Thermo Fisher Scientific
- Probe qPCR Mix
Comparison of resistance to inhibitory substances among PCR reagents
- Inhibitory effects caused by serial concentrations of humic, fulvic, and tannic acids in the standard mode real‐time PCR. Humic acid completely inhibited the reaction with GMM
- Tannic acid induced inhibition at the final concentration of 200 ng/µl for all the four reagents, where no amplification was observed for GMM and TMM
- GMM and FMM were more susceptible to PCR inhibition compared to the other three reagents in each of the standard and fast modes.
Comparison of inhibitor resistance of PCR reagents using field eDNA samples
- In the fast mode real‐time PCR, humic acid induced inhibition at the final concentration of 20 ng/µl for FMM and K3G with different magnitudes and it completely inhibited the reaction at the final concentration of 100 ng/µl for all the four reagents
- Tannic acid induced inhibition at the final concentration of 8 ng/µl for FMM, whereas no inhibition was observed for TMM and K3G until the final concentration of 200 ng/µl and for PQM until the final concentration of 40 ng/µl
References