Early identification of Aspergillus carbonarius in artificially and naturally contaminated grape berries by real-time polymerase chain reaction
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Keywords
Aspergillus carbonarius, detection, grapes, ochratoxin A, PCR
Abstract
Objectives The contamination of agricultural commodities with mycotoxins can occur without visible fungal contamination. Therefore, it is very important to develop a method for detecting very low levels of Aspergillus carbonarius DNA, which is the main agent responsible for ochratoxin A contamination in grape and wine. Methods The aim of this study was to detect the presence of A. carbonarius in grapes early during different stages of fungal development by the SYBRs Green real-time polymerase chain reaction or quantitative polymerase chain reaction approach by designing species-specific primers (Acpks) on the basis of the polyketide synthase sequences. Results The real-time polymerase chain reaction amplification results show early detection of a specific A. carbonarius from nonsymptomatic grape berries harvested directly from the field. Moreover, the amplification of fungal DNA extracted from grape berries artificially inoculated with A. carbonarius has shown an amplification starting from 6 h after inoculation, i.e. when the mycelium is not yet visible under stereomicroscope ( 100) observation. Conclusion The results indicate that these highly specific and sensitive polymerase chain reaction-based methods are able to discriminate grape berries infected with A. carbonarius from uninfected ones. Thus, in a working day, it is possible to harvest the berries, extract high-quality DNA and perform A. carbonarius-specific real-time polymerase chain reaction amplification. The method proposed in this work could contribute toward food safety (e.g., wine and table grapes) because it could be used to predict the potential risk of contamination by potentially ochratoxigenic strains of A. carbonarius.
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