Specific detection of Aspergillus parasiticus in wheat flour using a highly sensitive PCR assay

Abstract

Aspergillus parasiticus is one of the most important aflatoxin-producing species that contaminates foodstuffs and beverages for human consumption. In this work, a specific and highly sensitive PCR protocol was developed to detect A. parasiticus using primers designed on the multicopy internal transcribed region of the rDNA unit (ITS1-5.8S-ITS2 rDNA). The assay proved to be highly specific for A. parasiticus when tested on a wide range of related and other fungal species commonly found in commodities, and allowing discrimination from the closely related A. flavus. Accuracy of detection and quantification by conventional PCR were tested with genomic DNA obtained from wheat flour artificially contaminated with spore suspensions of known concentrations. Spore concentrations equal or higher than 10⁶ spore/g could be detected by the assay directly without prior incubation of the samples. The assay described is suitable for incorporation in routine analyses at critical points of the food chain within HACCP strategies.

Keywords:

• microbiology
• molecular biology
• PCR
• mycotoxins
• aflatoxins
• cereals

Acknowledgements

This work was supported by the Spanish MCyT (AGL 2007-66416-C05-02/ALI) and by the project UCM Santander Central Hispano GR58/08. J. Gil-Serna was supported by a FPU fellowship by the Spanish Ministry of Science and Innovation