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Mycotoxigenic Fungi pp 261–268Cite as

Multiplex Detection of Aspergillus Species

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1542))

Abstract

Multiplex real-time polymerase chain reaction (PCR) provides a fast and accurate DNA-based tool for the simultaneous amplification of more than one target sequence in a single reaction. Here a duplex real-time PCR assay is described for the simultaneous detection of Aspergillus carbonarius and members of the Aspergillus niger aggregate, which are the main responsible species for ochratoxin A (OTA) contamination in grapes. This single tube reaction targets the beta-ketosynthase and the acyl transferase domains of the polyketide synthase of A. carbonarius and the A. niger aggregate, respectively.

Besides, a rapid and efficient fungi DNA extraction procedure is described suitable to be applied in wine grapes. It includes a pulsifier equipment to remove conidia from grapes which prevents releasing of PCR inhibitors.

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References

  1. Battilani P, Magan N, Logrieco A (2006) European research on ochratoxinA in grapes and wine. Int J Food Microbiol 111:S2–S4

    Article  CAS  PubMed  Google Scholar 

  2. Perrone G, Susca A, Cozzi K et al (2007) Biodiversity of Aspergillus species in some important agricultural products. Stud Mycol 59:53–66

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Martínez-Culebras PV, Crespo Sempere A, Sánchez Hervás M et al (2009) Molecular characterization of the black Aspergillus isolates responsible for ochratoxin A contamination in grapes and wine in relation to taxonomy of Aspergillus section Nigri. Int J Food Microbiol 132:33–34

    Article  PubMed  Google Scholar 

  4. Abarca ML, Accensi F, Bragulat MR et al (2003) Aspergillus carbonarius as the main source of ochratoxin A contamination in dried vine fruits from the Spanish market. J Food Prot 66:504–506

    Article  CAS  PubMed  Google Scholar 

  5. Samson RA, Houbraken JAMP, Kuijpers AFA et al (2004) New ochratoxin A or sclerotium producing species in Aspergillus section Nigri. Stud Mycol 50:45–61

    Google Scholar 

  6. Bau M, Abarca ML, Bragulat MR et al (2005) Ochratoxigenic species from Spanish wine grapes. Int J Food Microbiol 98:125–130

    Article  CAS  PubMed  Google Scholar 

  7. Sage L, Krivobok S, Delbos E et al (2002) Fungal flora and ochratoxin A production in grapes and musts from France. J Agric Food Chem 50:1306–1311

    Article  CAS  PubMed  Google Scholar 

  8. Cabañes FJ, Accensi F, Bragulat MR et al (2002) What is the source of ochratoxin A in wine? Int J Food Microbiol 79:213–215

    Article  PubMed  Google Scholar 

  9. Battilani P, Pietri A (2002) Ochratoxin A in grapes and wine. Eur J Plant Pathol 108:639–643

    Article  CAS  Google Scholar 

  10. Bellí N, Pardo E, Marín S et al (2004) Occurrence of ochratoxin A and toxigenic potential of fungal isolates from Spanish grapes. J Sci Food Agric 84:541–546

    Article  Google Scholar 

  11. Martínez-Culebras PV, Ramón D (2007) An ITS-RFLP method to identify black Aspergillus isolates responsible for OTA contamination in grapes and wine. Int J Food Microbiol 113:147–153

    Article  PubMed  Google Scholar 

  12. Selma MV, Martínez-Culebras PV, Aznar R (2008) Real-time PCR based procedures for detection and quantification of Aspergillus carbonarius in wine grapes. Int J Food Microbiol 122:126–134

    Article  CAS  PubMed  Google Scholar 

  13. Selma MV, Martínez-Culebras PV, Elizaquível P et al (2009) Simultaneous detection of the main black aspergilli responsible for ochratoxin A (OTA) contamination in grapes by multiplex real-time polymerase chain reaction. Food Addit Contam 26:180–188

    Article  CAS  Google Scholar 

  14. Pitt JI, Hocking AD (1997) Fungi and food spoilage. Blackie Academic and Professional, London

    Book  Google Scholar 

  15. Cenis JL (1992) Rapid extraction of fungal DNA for PCR amplification. Nucleic Acids Res 20:2380

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Crespo-Sempere A, Martínez-Culebras PV, González-Candelas L (2014) The loss of the inducible Aspergillus carbonarius MFS transporter MfsA leads to ochratoxin A overproduction. Int J Food Microbiol 181:1–9

    Article  CAS  PubMed  Google Scholar 

  17. Higuchi R, Fockler C, Dollinger G et al (1993) Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. Biotechnology 11:1026–1030

    Article  CAS  PubMed  Google Scholar 

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Author information

Authors and Affiliations

  1. Department of Preventive Medicine, Public Health, Food Science and Technology, Bromatology, Toxicology, and Legal Medicine, University of Valencia, Valencia, Spain

    Pedro Martínez-Culebras

  2. Department of Biotechnology, Institute of Agrochemistry and Food Technology (IATA-CSIC), Valencia, Spain

    Pedro Martínez-Culebras & Rosa Aznar

  3. Research Group on Quality Safety and Bioactivity of Plant Foods, Department of Food Science and Technology, CEBAS-CSIC, Murcia, Spain

    María Victoria Selma

  4. Department of Microbiology and Ecology and Spanish Type Culture Collection (CECT), University of Valencia, Valencia, Spain

    Rosa Aznar

Authors
  1. Pedro Martínez-Culebras
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  2. María Victoria Selma
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  3. Rosa Aznar
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Corresponding author

Correspondence to Rosa Aznar .

Editor information

Editors and Affiliations

  1. Institute of Sciences of Food Production National Research Council, Bari, Italy

    Antonio Moretti

  2. Institute of Sciences of Food Production National Research Council, Bari, Italy

    Antonia Susca

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Martínez-Culebras, P., Selma, M.V., Aznar, R. (2017). Multiplex Detection of Aspergillus Species. In: Moretti, A., Susca, A. (eds) Mycotoxigenic Fungi. Methods in Molecular Biology, vol 1542. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6707-0_17

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  • DOI: https://doi.org/10.1007/978-1-4939-6707-0_17

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6705-6

  • Online ISBN: 978-1-4939-6707-0

  • eBook Packages: Springer Protocols

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