Development of Lab-on-a-Chip LAMP and Real-Time PCR Assays to Detect Aflatoxigenic Aspergillus flavus and Aspergillus parasiticus in Hazelnuts

Aflatoxins, which are potentially genotoxic and carcinogenic substances, are mainly produced by the <i>Aspergillus</i> section <i>Flavi</i>, including <i>Aspergillus flavus</i> and <i>A. parasiticus</i>. Current <i>Aspergillus</i> spp. detection is often based on molecular methods, such as real-time PCR and loop-mediated isothermal amplification (LAMP), targeting genes of the aflatoxin biosynthetic cluster. In this study, we developed a Lab-on-a-Chip (LoC) method based on real-time PCR and on LAMP for the specific detection of aflatoxigenic strains of <i>A. flavus</i> and <i>A. parasiticus</i> from infected hazelnuts. LoC-LAMP and LoC-real-time PCR assays were tested in terms of specificity, sensitivity, speed, and repeatability. The microfluidic chip allowed quick, specific, sensitive, simple, automatized, cheap, and user-friendly detection of aflatoxigenic strains of <i>A. flavus</i> and <i>A. parasiticus</i>. The LoC-LAMP showed a limit of detection (LOD) of 10 fg of DNA, while the LoC-real-time PCR showed a LOD of 10 pg of DNA. Achieving comparable sensitivity to that of LAMP and real-time PCR techniques, both LoC methods developed in this work offer the advantages of automation, minimal sample requirements, reagent requirements, and cost-effectiveness. Overall, the developed methods open the perspective for alternative monitoring of aflatoxigenic fungi in the agri-food industry.