Evaluation of Real‐Time PCR Amplification Efficiencies to Detect PCR Inhibitors

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Abstract
Real‐time PCR analysis is a sensitive template DNA quantitation strategy that has recently gained considerable attention in the forensic community. However, the utility of real‐time PCR methods extends beyond quantitation and allows for simultaneous evaluation of template DNA extraction quality. This study presents a computational method that allows analysts to identify problematic samples with statistical reliability by comparing the amplification efficiencies of unknown template DNA samples with clean standards. In this study, assays with varying concentrations of tannic acid are used to evaluate and adjust sample‐specific amplification efficiency calculation methods in order to optimize their inhibitor detection capabilities. Kinetic outlier detection and prediction boundaries are calculated to identify amplification efficiency outliers. Sample‐specific amplification efficiencies calculated over a four‐cycle interval starting at the threshold cycle can be used to detect reliably the presence of 0.4 ng of tannic acid in a 25 μL PCR reaction. This approach provides analysts with a precise measure of inhibition severity when template samples are compromised. Early detection of problematic samples allows analysts the opportunity to consider inhibitor mitigation strategies prior to genotype or DNA sequence analysis, thereby facilitating sample processing in high‐throughput forensic operations.