Food Analytical Methods

, Volume 7, Issue 10, pp 1950–1959 | Cite as

Comparison of DNA Extraction and PCR Setup Methods for Use in High-Throughput DNA Barcoding of Fish Species

  • Rosalee S. Hellberg
  • Michael D. Kawalek
  • Khanh T. Van
  • Yuelian Shen
  • Donna M. Williams-Hill
Article
First Online:
Received:
Accepted:

Abstract

DNA barcoding is a sequencing-based method that can be used for the identification of fish species in a regulatory setting. The objective of this study was to compare modified versions of three DNA extraction kits (i.e., Qiagen DNeasy Blood and Tissue Kit, Sigma-Aldrich Extract-N-Amp Kit; and Life Technologies MagMax-96 DNA Multi-Sample Kit) and two polymerase chain reaction (PCR) setup methods (manual vs. automated) for use in DNA barcoding, with a focus on minimizing time, costs, and labor. DNA was extracted from 83 fish products using each of the three kits and the results were compared based on sequencing success and sequencing quality parameters. A subset of 14 fish products was also tested in triplicate to compare PCR setup methods. Initially, reduced sequencing success was observed with the MagMax Kit (88 %) compared to the other two kits (95–96 %); however, after PCR and sequencing were repeated for DNA samples that initially failed, all three methods showed very high sequencing success (98–99 %). Overall, the modified Extract-N-Amp Kit offered the greatest reduction in time and costs, while the DNeasy Blood and Tissue Kit produced sequences with the highest quality and highest initial success rates. Automation of the PCR setup process resulted in slightly greater success (100 %) compared to manual PCR setup (98 %), and reduced the potential for human error that may result from manual pipetting. The results of this study demonstrate the advantages of incorporating rapid and/or automated methods into the DNA barcoding workflow, especially with regard to high-throughput operations.

Keywords

DNA barcoding DNA extraction Fish species identification PCR Sequencing 
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Notes

Acknowledgments

We thank the microbiology branch at the Food and Drug Administration (FDA) Pacific Regional Laboratory Southwest for setting aside fish tissue samples for this project. We also thank Jonathan R. Deeds and Sara M. Handy for critically reviewing the manuscript. This work was supported in part by the FDA Commissioner’s Fellowship Program.

Disclaimer

The views in this publication represent those of the authors. The inclusion of specific trade names or technologies does not imply endorsement by the U.S. FDA nor is criticism implied of similar commercial technologies not mentioned within.

Conflict of Interest

Rosalee Hellberg declares that she has no conflict of interest. Michael Kawalek declares that he has no conflict of interest. Khanh Van declares that he has no conflict of interest. Yuelian Shen declares that she has no conflict of interest. Donna Williams-Hill declares that she has no conflict of interest. This article does not contain any studies with human or animal subjects.

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Rosalee S. Hellberg
    • 1
  • Michael D. Kawalek
    • 2
  • Khanh T. Van
    • 2
  • Yuelian Shen
    • 3
  • Donna M. Williams-Hill
    • 2
  1. 1.School of Earth and Environmental Sciences, Food Science and NutritionChapman UniversityOrangeUSA
  2. 2.Office of Regulatory Affairs, Pacific Regional Laboratory Southwest, U.S. Food and Drug AdministrationIrvineUSA
  3. 3.Office of Regulatory Affairs, Office of Regional Operations/Division of Field Science, U.S. Food and Drug AdministrationRockvilleUSA

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