Early detection of ochratoxigenic fungi in wine grapes and of ochratoxin A in wine

The main objective of this work was to develop a protocol for the detection and quantification of ochratoxin A (OTA) to be used directly in food commodities, where sensitivity and robustness are critical factors. In this study, the contamination of wine grapes (var. Merlot) by OTA producer Aspergillus carbonarius was detected by PCR on asymptomatic grape berries. Four varieties of Vitis vinifera harvested directly from the field were used in the PCR-based assay. In the experiments, A. carbonarius was used to develop the PCR method, and Aspergillus niger strain 7096 and other species isolated from grapes were also used to test the specificity of the method presented below. The multiplex PCR reaction was tested on fungal DNA by amplifying two target genes in order to differentiate with an higher degree of effectiveness potentially ochratoxigenic black Aspergilli from other fungal contaminants. These PCR-based assays showed high sensitivity and effectiveness in detecting A. carbonarius from grape DNA. The same grape berries in which A. carbonarius DNA was revealed at an early timepoint showed the visible presence of black rot after appropriate incubation, confirming the results obtained by the molecular methods developed here. Furthermore, OTA detection on Merlot wine was performed with a novel device based on the use of an amorphous silica photosensor. This technique, using very simplified and rapid extraction procedures, allowed the detection and quantification of OTA from wine contaminated at 2 ppb. These methods allow rapid and early detection of the presence of the pathogen and of the toxin within a working day.