Ochratoxin A is a secondary metabolite with nephrotoxic and carcinogenic properties produced by several Aspergillus species. OTA is one of the main mycotoxin due to its high toxicity to humans and animals. Moreover, the maximum OTA limits allowed in food and raw agroproducts are under legal regulation. Aspergillus ochraceus is an important OTA producer species and it is considered the main source in coffee. Early detection of OTA producer species is a critical point to prevent that mycotoxin entering the food chain. Molecular techniques based in DNA are rapid, sensitive and specific and make possible to obtain relevant information on fungal strains. The aim of this work was to develop a method based in qPCR assays to detect A. ochraceus strains in two of its more common substrates: grapes and green coffee. Extraction of fungal and green coffee DNA was carried out with DNeasy Plant Mini Kit (QIAgen). This protocol was modified for DNA extraction from grapes adding a hydrosoluble polymer, polyvinylpirrolidone, in cell lyses and precipitation steps. qPCR reactions were performed using an ABI PRISM 7900HT Sequence Detection System and SYBR® Green PCR Master Mix (Applied Biosystems). The pair of primers used were located in ITS region and were specific for A. ochraceus strains. We have confirmed negative amplification with DNA of grapes and green coffee and that the presence of this exogenous DNA did not interfere in qPCR assay designed. This method was efficient to detect A. ochraceus strains in samples of grapes and green coffee artificially contaminated with a fungal spore suspension and using genomic DNA extracted after 0, 8, 16 and 24 hours of incubation. In conclusion, the results obtained in the present work indicate that this method is suitable to detect Aspergillus ochraceus in contaminated samples even without sample incubation. This work was supported by the Spanish Ministery of Education and Science (AGL2004-07549-C05-05 and AGL2007-66416-C05-02) and UCM-CM-961014.
Full conference title:
9th EUROPEAN CONFERENCE ON FUNGAL GENETICS
- ECFG 9th (2008)