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Food Analytical Methods

, Volume 7, Issue 4, pp 946–955 | Cite as

Development of a Simple and Cost-Effective Bead-Milling Method for DNA Extraction from Fish Muscles

  • Armani AndreaEmail author
  • Tinacci Lara
  • Xiong Xiong
  • Titarenko Evgeniya
  • Guidi Alessandra
  • Castigliego Lorenzo
Article

Abstract

In the fish food sector, due to a growing globalization of the market, where intentional and unintentional frauds reach alarming levels, the molecular analysis is increasingly used by both official agencies, to enforce the law on traceability, and private companies, to verify the quality of goods. DNA extraction represents a necessary and critical step for all types of DNA analysis. Among the drawbacks associated with this procedure, there are handling of toxic materials, low DNA yield, and low throughput, due to time-consuming manual procedures. In this work, to overcome some of these problems, we developed an alternative method based on a bead-milling procedure without proteinase K digestion. The new method was then compared with both a salting-out protocol, developed in a previous work, and a commercial kit. Yield, spectrophotometric purity, electrophoretic degradation pattern, and amplificability of the extracted DNA were assessed. In particular, DNA amplificability was evaluated by comparing the band intensity on the gel, after amplification of the 16S rRNA and cytochrome oxidase I genes with a conventional PCR, and the take-off cycles, after amplification of the 16S rRNA gene with a real-time PCR. The results showed that the bead-based method allowed to obtain acceptable amounts of DNA, with good purity and good characteristics of amplificability. Although the salting-out method remains the most effective protocol in terms of pure performances, the bead-milling procedure can be considered a valid alternative, in the light of its lower demand in terms of labor and costs.

Keywords

DNA extraction Bead milling Fish muscle 

Notes

Conflict of Interest

Armani Andrea declares that he has no conflict of interest. Tinacci Lara declares that he has no conflict of interest. Xiong Xiong declares that he has no conflict of interest. Titarenko Evgeniya declares that he has no conflict of interest. Guidi Alessandra declares that he has no conflict of interest. Castigliego Lorenzo declares that he 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

  • Armani Andrea
    • 1
    Email author
  • Tinacci Lara
    • 1
  • Xiong Xiong
    • 1
  • Titarenko Evgeniya
    • 1
  • Guidi Alessandra
    • 1
  • Castigliego Lorenzo
    • 1
  1. 1.FishLab, Department of Veterinary SciencesUniversity of PisaPisaItaly

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