Differential susceptibility of PCR reactions to inhibitors: an important and unrecognised phenomenon

Abstract

BackgroundPCR inhibition by nucleic acid extracts is a well known yet poorly described phenomenon. Inhibition assessment generally depends on the assumption that inhibitors affect all PCR reactions to the same extent; i.e. that the reaction of interest and the control reaction are equally susceptible to inhibition. To test this assumption we performed inhibition assessment on DNA extracts from human urine samples, fresh urine and EDTA using different PCR reactions.ResultsWhen copurified inhibitors were assessed using two different PCR reactions one reaction appeared to be inhibited whilst the other was not. Further experiments using various concentrations of unextracted urine to inhibit six different PCR reactions revealed that susceptibility to inhibition was highly variable between reactions. Similar results were obtained using EDTA as the PCR inhibitor. We could find no obvious explanation why one reaction should be more susceptible to inhibition than another, although a possible association with amplicon GC content was noted.ConclusionThese findings have serious implications for all PCR-based gene expression studies, including the relatively new PCR array method, and for both qualitative and quantitative PCR-based molecular diagnostic assays, suggesting that careful consideration should be given to inhibition compatibility when conducting PCR analyses. We have demonstrated unequivocally that it is not safe to assume that different PCR reactions are equally susceptible to inhibition by substances co-purified in nucleic acid extracts.

The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments

“…Although such approaches such as spiking samples with positive controls (52 ) can be used to detect inhibition, different PCR reactions may not be equally susceptible to inhibition by substances copurified in nucleic acid extracts (56,57 ). Consequently, it is better to routinely use dilutions of nucleic acids to demonstrate that observed decreases in C q s or copy numbers are consistent with the anticipated result and to report these data.…”

The Digital MIQE Guidelines: Minimum Information for Publication of Quantitative Digital PCR Experiments

“…dPCR is not dependent on amplification curves that may be affected by subtle inhibitors as in qPCR. However, dPCR remains susceptible to gross inhibitors that completely inhibit the reaction, which can be both primer and reagent-specific (36 ). Control experiments (37 ) should be included to ensure that inhibitors are not causing undetected problems in dPCR, especially when a negative result is being reported.…”

A Sample Extraction Method for Faster, More Sensitive PCR-Based Detection of Pathogens in Blood Culture

“…[5][6][7][8][9][10][11] In addition to SPS, several blood components (including heme, hematin, hemoglobin, lactoferrin, and IgG) can interfere with PCR, as do the anticoagulants used during blood collection, such as heparin and EDTA. [12][13][14][15][16][17][18][19][20] These inhibitors must be sufficiently removed from a blood culture sample to minimize the potential for false-negative PCR results.…”