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Development of a Protocol for Efficient DNA Extraction of Patulin-Producing Molds from Food for Sensitive Detection by PCR

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Abstract

Early detection of patulin-producing molds in foodstuffs is very important for food industry and for ensuring the consumers’ health. For precise and sensitive detection of patulin-producing molds by polymerase chain reaction (PCR) protocol, a previously efficient DNA extraction from foods is necessary, since variations in the efficiency can affect the detectability. In this study, eight different DNA extraction methods including a wide variety of treatments were assayed and compared in order to select the most efficient. Methods which combined thermal conidia breakdown and DNA commercial extraction kits reached higher yields, obtaining more quantity and quality in DNA extraction than those methods without extraction kit. In the present work, a simple method for fungal DNA extraction which is relatively rapid by means of minimizing the use of costly chemicals such as sodium dodecyl sulfate, mercaptoethanol, etc., or dangerous such as phenol/chlorophorm, was developed. When the detection limits were evaluated after PCR amplification using DNA from nine inoculated food matrices with the eight methods tested, the method including thermal conidia breakdown, extraction with cetyl trimethylammonium bromide/phosphate-buffered saline (1:2) buffer, and E.Z.N.A. Fungal DNA Mini Kit was chosen as the most adequate for detecting patulin-producing molds by PCR. This method allowed detection limits ranged from 102–103 conidia/g.

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Acknowledgements

This work has been funded by project AGL2007-64639 by the Spanish Comision Interministerial de Ciencia y Tecnología, Carnisenusa CSD2007-00016, Consolider Ingenio 2010, and GRU08100 and GRU09158 of the Junta of Extremadura and FEDER. M. Isabel Luque was the recipient of a contract from the Technician formation program from the Spanish Ministerio de Educación y Ciencia.

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  1. Higiene y Seguridad Alimentaria. Facultad de Veterinaria, Universidad de Extremadura, Avda. de la Universidad s/n, 10003, Cáceres, Spain

    M. Isabel Luque, María J. Andrade, Alicia Rodríguez, Mar Rodríguez & Juan J. Córdoba

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  1. M. Isabel Luque
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  2. María J. Andrade
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  3. Alicia Rodríguez
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  4. Mar Rodríguez
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  5. Juan J. Córdoba
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Correspondence to Juan J. Córdoba.

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Luque, M.I., Andrade, M.J., Rodríguez, A. et al. Development of a Protocol for Efficient DNA Extraction of Patulin-Producing Molds from Food for Sensitive Detection by PCR. Food Anal. Methods 5, 684–694 (2012). https://doi.org/10.1007/s12161-011-9298-x

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