Journal
INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY
Volume 131, Issue 2-3, Pages 162-167Publisher
ELSEVIER
DOI: 10.1016/j.ijfoodmicro.2009.02.008
Keywords
Aspergillus ochraceus; Aspergillus westerdijkiae; Real-time qPCR; OTA; Grapes; Green coffee beans; ITS
Categories
Funding
- Spanish Ministry of Science and Innovation [AGL 2007-66416-C05-02/ALI, CCG07-UCM/AGR-2612]
- FPU fellowship by the Spanish Ministry of Science and Innovation
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Aspergillus ochraceus and A. westerdijkiae are considered the most important Ochratoxin A (oTA) producing species included in Aspergillus section Circumdati which contaminate foodstuffs and beverages for human consumption. In this work a real-time quantitative PCR protocol was developed to detect both species using SYBR Green and primers designed on the basis of the multicopy ITS1 region of the rDNA. The assay had high efficiency (94%) and showed no inhibition by host or fungal DNA other than the target species. The lower detection limit of the target DNA was 2.5 pg/reaction. Accuracy of detection and quantification by qPCR were tested with genomic DNA obtained from green coffee beans and grapes artificially contaminated with spore suspensions of known concentrations. Spore concentrations equal or higher than 10(6) spore/ml could be detected by the assay directly without prior incubation of the samples and a positive relationship was observed between incubation time and qPCR values. The assay developed would allow rapid, specific, accurate and sensitive detection and quantification of A. ochraceus and A. westerdijkiae to be directly used in a critical point off the food chain, before harvesting green coffee and grape berries, to predict and control fungal growth and OTA production. (C) 2009 Elsevier B.V. All rights reserved.
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