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) Investigator® Quantiplex® Pro Kit, (2) Quantifiler® Trio DNA Quantification Kit, (3) PowerQuant® System, and (4) InnoQuant® 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 Investigator® Quantiplex® Pro Kit was the most tolerant kit to inhibitors and provided the most accurate quantification results with higher concentrations of inhibitors (except with salt). PowerQuant® and InnoQuant® 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 Investigator® Quantiplex® Pro indicating the largest DI and Quantifiler® Trio indicating the smallest DI. When the qPCR kits were paired with their respective STR kit to genotype highly degraded samples, the Investigator® 24plex QS and GlobalFiler® kits generated more complete profiles when the small target concentrations were used for calculating input amount.
KeywordsForensic science DNA quantification Quantitative real-time PCR Degradation index PCR inhibition
The authors would like to thank QIAGEN, Thermo Fisher Scientific, Promega Corporation, and InnoGenomics Technologies for supplying some of the commercial qPCR kits used in this study. The authors would also like to thank the staff at the Southeast Texas Applied Forensic Science Facility (STAFS) at Sam Houston State University for their assistance, and the individuals and families of those who donated their bodies for scientific research.
Role of Funding
This study was partially funded by a Graduate Research Fellowship Award No. 2015-R2-CX-0029 (National Institute of Justice, Office of Justice Programs, U.S. Department of Justice).
The opinions, findings, conclusions, or recommendations expressed in this article are those of the authors and do not necessarily reflect those of the National Institute of Justice.
Compliance with ethical standards
For the degraded samples (bone, decomposed and formalin-damaged tissues) used in this study, Code 45 of US Federal Regulations part 46102(f) exempts the requirement for Institutional Review Board (IRB) approval regarding the use of human cadaveric samples. All procedures were in accordance with the 1964 Helsinki Declaration and its later amendments.
Conflict of interest
The authors declare that they have no competing interests.
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