Highly sensitive PCR-based detection method specific for Aspergillus flavus in wheat flour

Aspergillus flavus is frequently found in food, producing a wide variety of toxins, aflatoxins being the most relevant in food safety. A specific PCR-based protocol for this species is described which allowed discrimination from other closely related species having different profiles of secondary metabolites from the Aspergillus Section Flavi, particularly A. parasiticus. The specific primers were designed on the multi-copy internal transcribed region of the rDNA unit (ITS1-5.8S-ITS2 rDNA) and were tested in a wide sample of related species and other fungal species commonly found in food. The PCR assay was coupled with a fungal enrichment and a DNA extraction method for wheat flour to enhance the sensitivity of the diagnostic protocol. The results indicated that the critical PCR amplification product was clearly observed for wheat flour contaminated by 10(2) spores after 16 h of incubation.