Optimization of DNA extraction and application of qPCR and ddPCR assays for detection of toxigenic Aspergillus flavus in hazelnut kernels

Aflatoxin contamination in hazelnuts, primarily caused by Aspergillus flavus, poses significant risks to food safety and public health, requiring highly sensitive and robust toxin detection strategies. While conventional culturing techniques remain relevant, they are time-consuming and prone to misidentifications. A molecular workflow for early detection of aflatoxigenic A. flavus in hazelnuts was developed and validated, combining an optimized DNA extraction protocol with quantitative PCR (qPCR) and droplet digital PCR (ddPCR) assays. Four DNA extraction methods were compared for their DNA yields and purity. DNA extraction protocol was optimized introducing a Tween-80 separation step, and was tested on hazelnuts artificially inoculated with aflatoxigenic A. flavus conidia. The optimized protocol was then validated for naturally contaminated hazelnut samples to assess its practical applicability, and to benchmark the performance of qPCR and ddPCR on real samples. The optimized protocol gave yield, purity and amplifiability, and appeared more appropriate for detecting aflatoxigenic A. flavus DNA in complex food matrices such as hazelnuts. The qPCR and ddPCR protocols detected target DNA, with ddPCR offering enhanced sensitivity and superior analytical performance. The developed protocol showed an increased sensitivity and quantification precision compared with previously developed methods. This research provides a validated molecular workflow for the early and sensitive detection of A. flavus in hazelnuts, offering a tool for preventive food safety monitoring and supporting aflatoxin risk assessment strategies for the hazelnut value chain.