Multi-gene probing of aflatoxigenic molds in food using PCR and immunological techniques

Aflatoxins are carcinogenic metabolites produced by several members of the Aspergillus flavus group in grains and foods. Three genes: ver-1, omt-1 and apa-2, coding for key enzymes and a regulatory factor in aflatoxin biosynthesis respectively, have been identified and their DNA sequences published. In the present study, three primer pairs were generated, each complementing the coding portion of one of the genes. DNA extracted from mycelia of five Aspergillus species, four Penicillium species and two Fusarium species were used as PCR templates for each of the primer pairs. DNA extracted from peanut, corn and three insect species commonly found in stored grains was also tested. Positive results (DNA amplification) were achieved only with DNA of the aflatoxigenic molds A. parasiticus and A. flavus in all three primer pairs. The detection limit of the PCR was determined using the primer pairs complementing the omt-1 and ver-1 genes. Sterile corn flour was inoculated separately with six different molds, each at several spore concentrations. Positive results were only obtained following a 24-h incubation in enriched media, with extracts of corn inoculated with A. parasiticus or A. flavus, even at the lowest spore concentration applied (10² spores/g). No DNA amplification was observed from corn inoculated with other molds, even at the highest inoculum level (10⁶ spores/g).