2Department of Biology, McMaster University, Hamilton, ON, Canada
3Department of Pathology and Molecular Medicine, McMaster University, Hamilton, ON, Canada
Inhibition is problematic in many applications of PCR, particularly those involving degraded or low amounts of template DNA, when simply diluting the extract is undesirable. Two basic approaches to monitoring inhibition in such samples using real-time or quantitative PCR (qPCR) have been proposed. The first method analyzes the quantification cycle (Cq) deviation of a spiked internal positive control. The second method considers variations in reaction efficiency based on the slopes of individual amplification plots. In combining these methods, we observed increased Cq values together with reduced amplification efficiencies in some samples, as expected; however, deviations from this pattern in other samples support the use of both measurements. Repeat inhibition testing enables optimization of PCR facilitator combinations and sample dilution such that DNA yields and/or quantitative accuracy can be maximized in subsequent PCR runs. Although some trends were apparent within sample types, differences in inhibition levels, optimal reactions conditions, and expected recovery of DNA under these conditions suggest that all samples be routinely tested with this approach.
Since the advent of PCR, many inhibitory substances that interfere with the activity or availability of particular reaction components have been identified (1,2). The presence of inhibitors introduces a number of problems, ranging from reduced amplification capacity and reduced assay sensitivity to complete reaction failure. Thus, in situations where high (or simply consistent) amplicon concentrations or maximal sensitivity are desired, or when drawing conclusions from negative results, it is important that PCR inhibition be detected and circumvented as much as possible. Inhibition of qPCR presents additional concerns, as slight variations in amplification efficiency between samples can drastically affect the accuracy of template quantification (3).
When PCR inhibition is suspected, the simplest course of action is to dilute the template (and inhibitors), and make use of the sensitivity of PCR (1); however, for applications involving heavily degraded or otherwise low-copy templates, this solution is often undesirable—and indeed sometimes impossible—due to the further reduction of template amounts. In these situations, which are commonly encountered in the forensic and ancient DNA fields, routine detection and quantification of PCR inhibition is necessary.
In standard PCR experiments, negative results or unexpectedly low product yields may be indicative of inhibition, provided that the template is known to be present; alternatively, a known amount of non-endogenous DNA can be added to the sample and amplified as an internal positive control (IPC). These controls may be used in qPCR assays as well, enabling quantitative assessments of their performance. Based on modeling individual reaction kinetics and/or the calculation of amplification efficiency, qPCR also allows inhibited samples to be identified without additional IPC amplifications.Internal positive controls
A number of studies have incorporated IPC assays for inhibition detection (4,5,6,7,8,9), as have some commercially available systems such as the Quantifiler Human DNA Quantification Kit (Applied Biosystems, Foster City, CA, USA). Typically, problematic samples are identified based on the shift in Cq (ΔCq) observed relative to an uninhibited reaction. When using this type of control, one assumes that the effects of inhibitors on the IPC are predictive of those on other targets in subsequent PCR assays from the same sample. This assumption has been challenged, as some authors have noted differential susceptibility to PCR inhibition between assays (10,11).
Huggett et al. (11) found quantitatively small but significant differences between assays, with no correlation between the extent of inhibition and any particular characteristic of the template or primer sequences. Ståhlberg et al. (10) hypothesize that this may result from indirect inhibition, through competition for particular reaction components. If, for example, Mg2+ ions became limiting by the presence of EDTA in a sample, an assay with a greater Mg2+ requirement would be more susceptible to the same level of EDTA inhibition.
Thus, it is important that both IPC and target reactions perform well under (ideally) the same conditions. In particular, the concentrations of components known to facilitate PCR in the presence of inhibitors (e.g., BSA, Taq polymerase, etc.) must be the same. Furthermore, IPC and target assays with different enzymatic requirements may not be comparable; for instance, if inhibition of the exonuclease activity of Taq is also measured (e.g., by a TaqMan IPC assay), target assays performed without such probes may be less susceptible to inhibition than the IPC predicts.Amplification efficiency
Based on the potential for differential susceptibility to inhibition, the Minimum Information for Publication of Quantitative Real-time PCR Experiments (MIQE) guidelines (12) instead advocate measuring amplification performance using a dilution series of each sample [e.g., Ståhlberg et al. (10)]. As noted previously, this may not be feasible for low-copy templates.
Building on the 2002 publications of Liu and Saint (13,14), various mathematical models that describe individual reaction kinetics have been developed and compared (3, 15,16,17,18,19,20,21,22,23,24,25). Regardless of the particular model, it is clear that kinetic outliers can be identified when inhibitors influence amplification efficiency; however, certain inhibitory mechanisms may not be identified this way (20). Furthermore, highly inhibited samples cannot be differentiated from those having no template, or from a combination of low-copy template and moderate inhibition when there is simply no amplification observed.