Evaluation of Four Commercial Quantitative Real-Time PCR Kits With Inhibited and Degraded Samples

• NCJ Number: 253261
• Journal: International Journal of Legal Medicine Volume: 132 Issue: 3 Dated: 2018 Pages: 691-701
• Author(s): Amy S. Holmes; Rachel Houston; Kyleen Elwick; David Gangitano; Sheree Hughes-Stamm
• Date Published: 2018
• Length: 11 pages
• Grant Number(s): 2015-R2-CX-0029
• Agencies: NIJ-Sponsored
• Publication Type: Research (Applied/Empirical); Report (Study/Research); Report (Grant Sponsored); Program/Project Evaluation; Program/Project Description
• Award: Improved sample collection and preservation of DNA from decomposing human remains; A direct PCR approach for faster disaster victim identification (DVI)

Annotation

Since 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, the current study compared 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.

Abstract

DNA quantification is a vital step in forensic DNA analysis to determine the optimal input amount for DNA typing. The results of the current 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. (publisher abstract modified)