Evaluation of four commercial quantitative real-time PCR kits with inhibited and degraded samples

DNA quantification is a vital step in forensic DNA analysis to determine the optimal input amount for DNA typing. A quantitative real-time polymerase chain reaction (qPCR) assay that can predict DNA degradation or inhibitors present in the sample prior to DNA amplification could aid forensic laboratories in creating a more streamlined and efficient workflow. This study compares the results from four commercial qPCR kits: (1) InvestigatorA (R) QuantiplexA (R) Pro Kit, (2) QuantifilerA (R) Trio DNA Quantification Kit, (3) PowerQuantA (R) System, and (4) InnoQuantA (R) HY with high molecular weight DNA, low template samples, degraded samples, and DNA spiked with various inhibitors.The results of this study indicate that all kits were comparable in accurately predicting quantities of high quality DNA down to the sub-picogram level. However, the InnoQuant(R) HY kit showed the highest precision across the DNA concentration range tested in this study. In addition, all kits performed similarly with low concentrations of forensically relevant PCR inhibitors. However, in general, the InvestigatorA (R) QuantiplexA (R) Pro Kit was the most tolerant kit to inhibitors and provided the most accurate quantification results with higher concentrations of inhibitors (except with salt). PowerQuantA (R) and InnoQuantA (R) HY were the most sensitive to inhibitors, but they did indicate significant levels of PCR inhibition. When quantifying degraded samples, each kit provided different degradation indices (DI), with InvestigatorA (R) QuantiplexA (R) Pro indicating the largest DI and QuantifilerA (R) Trio indicating the smallest DI. When the qPCR kits were paired with their respective STR kit to genotype highly degraded samples, the InvestigatorA (R) 24plex QS and GlobalFilerA (R) kits generated more complete profiles when the small target concentrations were used for calculating input amount.