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International Journal of Legal Medicine

, Volume 132, Issue 3, pp 691–701 | Cite as

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

  • Amy S. Holmes
  • Rachel Houston
  • Kyleen Elwick
  • David Gangitano
  • Sheree Hughes-Stamm
Original Article

Abstract

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.

Keywords

Forensic science DNA quantification Quantitative real-time PCR Degradation index PCR inhibition 

Notes

Acknowledgments

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.

Supplementary material

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References

  1. 1.
  2. 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. 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. 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. 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. 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. 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
  8. 8.
    Vernarecci S, Ottaviani E, Agostino A, Mei E, Calandro L, Montagna P (2015) Quantifiler® trio kit and forensic samples management: a matter of degradation. Forensic Sci Int Genet 16:77–85.  https://doi.org/10.1016/j.fsigen.2014.12.005 CrossRefPubMedGoogle Scholar
  9. 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
  10. 10.
    van den Berge M, Wiskerke D, Gerretsen RRR, Tabak J, Sijen T (2016) DNA and RNA profiling of excavated human remains with varying postmortem intervals. Int J Legal Med 130(6):1471–1480.  https://doi.org/10.1007/s00414-016-1438-9 CrossRefPubMedGoogle Scholar
  11. 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
  12. 12.
    Gettings KB, Kiesler KM, Vallone PM (2015) Performance of a next generation sequencing SNP assay on degraded DNA. Forensic Sci Int Genet 19:1–9.  https://doi.org/10.1016/j.fsigen.2015.04.010 CrossRefPubMedGoogle Scholar
  13. 13.
    Thompson RE, Duncan G, McCord BR (2014) An investigation of PCR inhibition using Plexor®-based quantitative PCR and short tandem repeat amplification. J Forensic Sci 59(6):1517–1529.  https://doi.org/10.1111/1556-4029.12556 CrossRefPubMedGoogle Scholar
  14. 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
  15. 15.
    Sorensen A, Berry C, Bruce D, Gahan ME, Hughes-Stamm S, McNevin D (2016) Direct-to-PCR tissue preservation for DNA profiling. Int J Of Legal Med 130(3):607–613.  https://doi.org/10.1007/s00414-015-1286-z CrossRefGoogle Scholar
  16. 16.
    Investigator(R) Quantiplex(R) Pro Handbook (April 2017)Google Scholar
  17. 17.
    Quantifiler(R) HP and Trio DNA Quantification Kits User Guide (2017)Google Scholar
  18. 18.
    PowerQuant System Technical Manual (March 2015)Google Scholar
  19. 19.
    InnoQuant(R) HY User Guide v1.5 (Dec. 2016)Google Scholar
  20. 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. 21.
    QIAamp(R) DNA Investigator Handbook (June 2012)Google Scholar
  22. 22.
    PrepFiler(R) and PrepFiler(R) BTA Forensic DNA Extraction Kits User Guide (2012)Google Scholar
  23. 23.
    Isolation of DNA from forensic casework samples part B (purification) protocol sheet, version 2 (May 2008)Google Scholar
  24. 24.
    QIAamp(R) DNA FFPE Tissue Handbook (June 2012)Google Scholar
  25. 25.
    Investigator(R) 24plex QS Handbook (July 2016)Google Scholar
  26. 26.
    GlobalFiler™ PCR Amplification kit user guide (2016)Google Scholar
  27. 27.
    PowerPlex(R) Fusion 6C System for Use on the Applied Biosystems (R) Genetic Analyzers Technical Manual (April 2017)Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2017

Authors and Affiliations

  • Amy S. Holmes
    • 1
  • Rachel Houston
    • 1
  • Kyleen Elwick
    • 1
  • David Gangitano
    • 1
  • Sheree Hughes-Stamm
    • 1
  1. 1.Department of Forensic Science, College of Criminal JusticeSam Houston State UniversityHuntsvilleUSA

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