Polymerase chain reaction-based assay for the early detection of aflatoxigenic fungi on maize kernels

Abstract

Objectives The aim of this work was to set up a DNA-based method for detecting aflatoxigenic fungi Aspergillus flavus and Aspergillus parasiticus on whole maize kernels using species-specific primers. Methods In the experiments, two aflatoxigenic strains, A. flavus NRRL 3357 and A. parasiticus NRRL 2999, were used. Further, Aspergillus niger 7096, OTA producer strain, and other strains of Aspergillus spp., Penicillium spp. and Fusarium spp. isolated from maize kernels were analyzed. Twelve commercial maize hybrids employed in the food and feed industries, not inoculated and inoculated with the different fungi, at different water activities, were used in the polymerase chain reaction-based assay. Three primer pairs were used for amplifying fragments of Afl P (omt 1), Afl M (ver 1), Afl R, genes present in the aflatoxin biosynthesis cluster. Results DNA amplification was achieved only with DNA from fungal strains of A. parasiticus and A. flavus and from maize kernels inoculated with A. flavus or A. parasiticus. Amplification was never observed with DNA of the other fungal species. Amplification was evident in maize starting from 12 h of incubation after inoculation, when mycelium is not yet visible by stereomicroscope analysis. Conclusion The results indicate that these highly specific and sensitive polymerase chain reaction-based methods are able to discriminate maize kernels infected with A. flavus or A. parasiticus from uninfected ones. This polymerase chain reaction method could be a real, effective alternative to traditional diagnostic methods for the early detection of aflatoxigenic fungi in food commodities.