AcOTApks gene-based molecular tools to improve quantitative detection of the mycotoxigenic fungus Aspergillus carbonarius

Aspergillus carbonarius is the main producer of Ochratoxin A (OTA) in grape, a mycotoxin frequently detected in grapes and derivatives and classified as a possible human carcinogen by the IARC. Several molecular methods are available for A. carbonarius detection and quantification, although the correlation between the consistency of fungal population and OTA contamination needs to be improved. This study aimed to develop new quantitative PCR (qPCR) and digital droplet PCR (ddPCR) to quantify A. carbonarius OTA-producing strains based on the key gene AcOTApks (otaA). Different primers/probe sets targeting the AcOTApks gene were obtained and validated for their specificity in silico and then using the gDNA of different OTA-producing Aspergillus spp. and a panel of bacteria and fungi commonly associated with grapes. The method allowed to quantify up to 100 fg μL-1 (Cq=37) and 10 fg μL-1 (0.38 copies∙μL-1) of gDNA extracted from A. carbonarius mycelium in qPCR and ddPCR, respectively. The sensitivity with artificially contaminated must samples was up to 100 conidia (Cq=38) and 1 conidium (0.13 copies∙μL-1), with qPCR and ddPCR, respectively. The methods were validated on naturally infected grape must samples, and the quantification of the fungus, in both cases, highly correlated (r= 0.8) with OTA amount in the samples. In conclusion, we report new molecular diagnostic methods in ddPCR and qPCR, targeting the AcOTApks gene, which can be helpful in the management of OTA contamination in grapes and derivatives to ensure wine safety with a sustainable approach.