In this study, we developed a novel internal amplification control (IAC) that allows highly accurate adjustment of quantitative data for partial PCR inhibition. The extremely precise IAC approach described in this study allowed a highly accurate quantification of microorganisms in environmental soil DNA samples that contained substances that partially inhibited PCR amplification.
Environmental soil samples are commonly known to contain variable levels of PCR-inhibiting compounds (30
). These compounds are often coextracted with soil DNA during nucleic acid extraction procedures. A commonly applied strategy to reduce inhibition of PCR is to simply dilute soil DNA extracts until PCR inhibition is no longer observed. However, also the targets of interest are diluted, which negatively influences detection sensitivity. Additionally, this commonly used strategy (dilution series preparation) is not well suited for use with the new-generation qPCR platforms (15
), which require relatively high concentrations of DNA target input in nanoliter reaction volumes. Indeed, we observed that the reproducibility, accuracy, and precision of quantification in the Biotrove OpenArray PCR all decrease at lower input DNA copy numbers, stressing the need for concentrated DNA samples (Fig. ).
In order to test the developed IAC system, we induced PCR inhibition by adding ethanol and humic acids to the IAC samples at a range of concentrations. Addition of ethanol up to even a 5% final concentration did not result in any PCR inhibition in the Biotrove OpenArray system (data not shown). Higher ethanol concentrations were not expected or realistic based on typical DNA extraction protocols and were therefore not tested. PCR inhibition with humic acids, however, did result in significantly different sample IAC regression curve intercepts compared to the intercept of the calibration IAC regression curve. Since the slopes did not appear to be significantly different, artificially induced partial PCR inhibition resulted in a linear CT shift of all IAC concentrations. Thus, partial PCR inhibition in terms of CT values was not target concentration dependent in these samples. Analyzing the number of negative PCR amplifications at very high humic acid concentrations, however, indicated that full PCR inhibition in the Biotrove OpenArray system is more likely to occur at low target concentrations. This suggests that, in cases of very strong inhibition at low IAC concentrations, which result in negative PCR amplifications, a DNA sample might still have to be diluted to allow accurate IAC-based quantification. Such strong PCR inhibition was, however, only observed in the artificially inhibited PCR amplifications at very high humic acid concentrations and not in the analyzed DNA extracts from actual environmental samples (see below).
The developed calibration IAC regression formula was tested with DNA extracts from six marine clay and sandy soil environments, which contained various amounts of organic matter (19
). The relative abundances of three bacterial groups (the alpha and beta subdivisions of proteobacteria and actinobacteria) and the density of total bacteria in the soils were determined, while the potential PCR inhibition was monitored with the newly developed IAC. As expected, marine clay soils with high organic matter contents showed the highest degree of PCR inhibition, while sandy soils with low organic matter contents generally showed lower PCR inhibition. Contrary to those of the samples where PCR inhibition was induced artificially, the slopes of the sample IAC curves obtained with the PCR-inhibiting environmental samples were often different from the slope of the calibration IAC curve. Thus, partial PCR inhibition in environmental samples can be target concentration dependent, stressing the need for IACs at multiple target concentrations for more precise quantification (see Appendix S2 in the supplemental material). By comparing CT
values obtained from diluted DNA extracts with the IAC-corrected CT
values obtained from the inhibiting DNA extracts, we demonstrated that target-specific CT
values could be accurately adjusted for partial PCR inhibition with the developed IAC. It should be noted that the IAC primer pairs have no relation to the primer pairs of the assayed targets and that each new target primer pair has to be tested for the PCR inhibition correction in combination with the IAC system before reliable IAC-based quantification can be performed with environmental samples.
In the field 6 soil DNA extract, the slope of the IAC sample regression curve remained significantly different from the slope of the calibration IAC regression curve, even after 40-fold dilution of the DNA sample. Interestingly, the undiluted and 40-fold-diluted DNA sample IAC regression formulas were highly similar, implying that 40-fold dilution was not sufficient to neutralize completely the negative effect of the undiluted DNA extract on the sample IAC regression curve (further dilutions were not tested because of too much loss of sensitivity). Nevertheless, this event did not affect the possibility of accurate quantification with this new IAC principle.
Next-generation qPCR platforms, like the Biotrove OpenArray system, allow parallel mass testing of environmental samples against a wide range of microorganisms. Reliable quantification requires amplification controls that can correct for partial PCR inhibition, as is often observed for environmental soil DNA extracts. We demonstrated that artificially induced partial PCR inhibition in the Biotrove OpenArray system is not target concentration dependent, while low target concentrations were shown to be more sensitive to full PCR inhibition. Partial PCR inhibition in environmental samples, however, did appear to be target concentration dependent. The developed IAC system can be easily adapted for incorporation into TaqMan probe-based detection assays, and the bacterial cells containing the IAC plasmids could potentially be used as extraction controls in future experiments (10
). In addition, the performance of DNA extraction methods can be evaluated. The described IAC system enables accurate adjustments to obtain quantitative data from environmental DNA extracts that possibly inhibit PCR and can be applied for routine screenings of samples from different environmental systems.