Development of a Multiplex PCR Method for the Detection of Patulin-, Ochratoxin A- and Aflatoxin-Producing Moulds in Foo
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Development of a Multiplex PCR Method for the Detection of Patulin-, Ochratoxin A- and Aflatoxin-Producing Moulds in Foods María I. Luque & María J. Andrade & Alicia Rodríguez & Elena Bermúdez & Juan J. Córdoba
Received: 28 July 2012 / Accepted: 25 September 2012 / Published online: 9 October 2012 # Springer Science+Business Media New York 2012
Abstract A multiplex PCR was developed for the simultaneous detection of patulin-, ochratoxin A-, and aflatoxin-producing moulds. The idh, otanpsPN and omt-1 genes involved in the patulin, ochratoxin A and aflatoxin biosynthesis, respectively, were used as target sequences in the multiplex PCR. The expected amplicons of 496, 373, and 289 bp for the corresponding primer pairs were detected in all tested toxigenic mould strains. These results were closely related to the mycotoxin detection by micellar electrokinetic capillary electrophoresis and high-performance liquid chromatography–mass spectrometry. The sensitivity of the method was tested on DNA from mould pure cultures and DNA from artificially contaminated food products (dry-fermented sausage “salchichón”, paprika, apple, wheat and peanut). The method was able to detect down to 1 ng of pure DNA from producing strains and from 103 to 104 cfu/g in inoculated foods. Inhibition from the food matrices was no detected in the PCR assay. The developed multiplex PCR protocol is a good alternative to traditional diagnostic methods for the simultaneous and early detection of different types of toxigenic moulds in foods. Keywords Toxigenic moulds . Multiplex PCR . Detection . Foods
M. I. Luque : M. J. Andrade (*) : A. Rodríguez : E. Bermúdez : J. J. Córdoba Higiene y Seguridad Alimentaria. Facultad de Veterinaria, Universidad de Extremadura, Avda. de la Universidad s/n., 10003 Cáceres, Spain e-mail: [email protected] URL: http://higiene.unex.es
Introduction Contamination of foods by toxigenic fungi is a serious risk for the consumer health because of their potential for producing mycotoxins. These secondary metabolites are carcinogenic, teratogenic and hepatotoxic compounds (Bacha et al. 2009; Beretta et al. 2000; Kotsonis et al. 2001). Mycotoxins have been found on a wide variety of foodstuffs such as nuts (Leong et al. 2010), corn (Mansfield et al. 2008), fruits (Toscani et al. 2007), meat products (Hussain et al. 2010) and spices (Hashem and Al Amri 2010). The most common mould species involved in the mycotoxin production in foodstuffs belong to Penicillium and Aspergillus genera (Bacha et al. 2009). Within these toxigenic moulds, those producers of patulin, ochratoxin A (OTA) and aflatoxins pose the highest risk in most foods. In order to prevent the presence of these toxic compounds in foods, the early detection of toxigenic mould contamination throughout food processing is necessary. Due to the fact that the available traditional and immunological methods for detecting toxigenic moulds are labor intensive and time-consuming (Del Fiore et al. 2010), polymerase chain reaction (PCR)-based methods could be good alternative to tra
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