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.
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.
- 1.Quality Assurance Standards For Forensic DNA Testing Laboratories https://www.fbi.gov/file-repository/quality-assurance-standards-for-forensic-dna-testing-laboratories.pdf
- 2.Di Pasquale F, Cornelius S, König M, Scherer M, Schmid C, Dienemann C, Bochmann L, Prochnow A, Schnibbe T, Engel H (2011) Investigator® Quantiplex kit: for reliable quantification of human DNA in forensic samples. Forensic Sci Int Genet Suppl Ser 3(1):e413–e414. https://doi.org/10.1016/j.fsigss.2011.09.068
- 3.Loftus A, Murphy G, Brown H, Montgomery A, Tabak J, Baus J, Carroll M, Green A, Sikka S, Sinha S (2017) Development and validation of InnoQuant® HY, a system for quantitation and quality assessment of total human and male DNA using high copy targets. Forensic Sci Int Genet 29:205–217. https://doi.org/10.1016/j.fsigen.2017.04.009
- 4.Pineda GM, Montgomery AH, Thompson R, Indest B, Carroll M, Sinha SK (2014) Development and validation of InnoQuant™, a sensitive human DNA quantitation and degradation assessment method for forensic samples using high copy number mobile elements Alu and SVA. Forensic Sci Int Genet 13:224–235. https://doi.org/10.1016/j.fsigen.2014.08.007
- 5.Di Pasquale F, Cornelius S, König M, Scherer M, Prochnow A, Peist R (2015) Analysis and interpretation of difficult samples—the interaction of a DNA quantification kit with the right STR assay may facilitate processing of critical trace material. Forensic Sci Int Genet Suppl Ser 5:e407–e408. https://doi.org/10.1016/j.fsigss.2015.09.161
- 6.Ewing MM, Thompson JM, McLaren RS, Purpero VM, Thomas KJ, Dobrowski PA, DeGroot GA, Romsos EL, Storts DR (2016) Human DNA quantification and sample quality assessment: developmental validation of the PowerQuant® system. Forensic Sci Int Genet 23:166–177. https://doi.org/10.1016/j.fsigen.2016.04.007
- 7.Holt A, Wootton SC, Mulero JJ, Brzoska PM, Langit E, Green RL (2016) Developmental validation of the Quantifiler® HP and trio kits for human DNA quantification in forensic samples. Forensic Sci Int Genet 21:145–157. https://doi.org/10.1016/j.fsigen.2015.12.007
- 9.Goecker ZC, Swiontek SE, Lakhtakia A, Roy R (2016) Comparison of Quantifiler® trio and InnoQuant™ human DNA quantification kits for detection of DNA degradation in developed and aged fingerprints. Forensic Sci Int 263:132–138. https://doi.org/10.1016/j.forsciint.2016.04.009
- 11.Alaeddini R, Walsh SJ, Abbas A (2010) Forensic implications of genetic analyses from degraded DNA—a review. Forensic Sci Int Genet 4(3):148–157. https://doi.org/10.1016/j.fsigen.2009.09.007Google Scholar
- 14.Alaeddini R (2012) Forensic implications of PCR inhibition—a review. Forensic Sci Int Genet 6(3):297–305. https://doi.org/10.1016/j.fsigen.2011.08.006Google Scholar
- 16.Investigator(R) Quantiplex(R) Pro Handbook (April 2017)Google Scholar
- 17.Quantifiler(R) HP and Trio DNA Quantification Kits User Guide (2017)Google Scholar
- 18.PowerQuant System Technical Manual (March 2015)Google Scholar
- 19.InnoQuant(R) HY User Guide v1.5 (Dec. 2016)Google Scholar
- 20.National Institute of Standards & Technology Certificte of Analysis Standard Reference Material (R) 2372 Human DNA Quantification Standard, Issued 1/13 (Jan. 2013)Google Scholar
- 21.QIAamp(R) DNA Investigator Handbook (June 2012)Google Scholar
- 22.PrepFiler(R) and PrepFiler(R) BTA Forensic DNA Extraction Kits User Guide (2012)Google Scholar
- 23.Isolation of DNA from forensic casework samples part B (purification) protocol sheet, version 2 (May 2008)Google Scholar
- 24.QIAamp(R) DNA FFPE Tissue Handbook (June 2012)Google Scholar
- 25.Investigator(R) 24plex QS Handbook (July 2016)Google Scholar
- 26.GlobalFiler™ PCR Amplification kit user guide (2016)Google Scholar
- 27.PowerPlex(R) Fusion 6C System for Use on the Applied Biosystems (R) Genetic Analyzers Technical Manual (April 2017)Google Scholar