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

## TL;DR

This paper introduces a new Lab-on-a-Chip method to quickly and accurately detect harmful fungi in hazelnuts that produce cancer-causing aflatoxins.

## Contribution

A novel Lab-on-a-Chip system combining LAMP and real-time PCR for automated, cost-effective detection of aflatoxigenic fungi in hazelnuts.

## Key findings

- The LoC-LAMP method detected as little as 10 fg of DNA from aflatoxigenic fungi.
- The LoC-real-time PCR method had a detection limit of 10 pg of DNA.
- Both methods are automated, require minimal samples, and are cost-effective.

## Abstract

Aflatoxins, which are potentially genotoxic and carcinogenic substances, are mainly produced by the Aspergillus section Flavi, including Aspergillus flavus and A. parasiticus. Current Aspergillus 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 A. flavus and A. parasiticus 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 A. flavus and A. parasiticus. 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.