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Simultaneous Identification of Pork and Poultry Origins in Pet Foods by a Quick Multiplex Real-Time PCR Assay Using EvaGreen Florescence Dye

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Abstract

EvaGreen multiplex real-time polymerase chain reaction (EMRT-PCR) was designed for an assay that can join the advantages of multiplex PCR and real-time PCR to recognize animal genes more quickly in pet foods. EMRT-PCR based on melting temperatures discrimination by using EvaGreen fluorescence dye was developed for the analysis of pork and poultry in pet food. The method combines the use of poultry- and pork-specific primers that amplify small fragments of 12S rRNA and mitochondrial DNA genes. Appropriate mixtures of poultry and pork meat in reference samples were used to develop the assay. Gene yields of poultry and pork were represented in two melting peaks generated simultaneously at temperatures of 80.5 and 87.2 °C, respectively. Based upon the assay results, it has been concluded that EMRT-PCR assay might be an efficient tool for the verification of species origin in pet foods.

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Abbreviations

EMRT-PCR:

EvaGreen multiplex real-time polymerase chain reaction

Tm:

Melting temperature

GC:

The number of G's and C's in the primer

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Acknowledgments

The authors would like to thank the Office of Scientific Research Projects at Fatih University for supporting this study through project grant no. P50091102_G (1884) and the Department of Genetics and Bioengineering for providing the necessary facilities required for the study.

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Authors and Affiliations

  1. Department of Genetics and Bioengineering, Faculty of Engineering, Fatih University, Büyükçekmece, 34500, Istanbul, Turkey

    M. Safdar & M. F. Abasıyanık

Authors
  1. M. Safdar
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  2. M. F. Abasıyanık
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Corresponding author

Correspondence to M. Safdar.

Additional information

The paper highlights the following points.

• The first report of development of multiplex real-time PCR based on EvaGreen florescence dye for pork and poultry species detection simultaneously in pet foods.

• We report the assay which saves cost, time, chemicals, and materials because EvaGreen dye is cheaper than probe-based methods.

• We report the specificity and sensitivity of the assay has been evaluated very high as before reported.

• We report the developed system was successfully applied to commercial pet foods for detection of low quality meat species (poultry and pork).

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Safdar, M., Abasıyanık, M.F. Simultaneous Identification of Pork and Poultry Origins in Pet Foods by a Quick Multiplex Real-Time PCR Assay Using EvaGreen Florescence Dye. Appl Biochem Biotechnol 171, 1855–1864 (2013). https://doi.org/10.1007/s12010-013-0485-7

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  • DOI: https://doi.org/10.1007/s12010-013-0485-7

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