Detection and quantification of Aspergillus section Flavi spp. in stored peanuts by real-time PCR of nor-1 gene, and effects of storage conditions on aflatoxin production

Abstract

Aspergillus flavus and A. parasiticus are the main species from section Flavi responsible for aflatoxin accumulation in stored peanuts. A real-time PCR (RT-PCR) system directed against the nor-1 gene of the aflatoxin biosynthetic pathway as target sequence was applied to monitor and quantify Aspergillus section Flavi population in peanuts. Kernels were conditioned at four water activity ( a W) levels and stored during a 4-month period. The quantification of fungal genomic DNA in naturally contaminated peanut samples was performed using TaqMan fluorescent probe technology. Sensitivity tests demonstrated that DNA amounts accounting for a single conidium of A. parasiticus RCP08300 can be detected. A standard curve relating nor-1 copy numbers to colony forming units (cfu) was constructed. Counts of species of Aspergillus section Flavi from unknown samples obtained by molecular and conventional count (CC) methodologies were compared. A correlation between cfu data obtained by RT-PCR and CC methods was observed ( r = 0.613; p < 0.0001); and the former always showed values higher by 0.5–1 log units. A decrease of fungal density was observed throughout the storage period, regardless of the quantification methodology applied. Total aflatoxin levels ranging from 1.1 to 200.4 ng/g were registered in peanuts conditioned at the higher a W values (0.94–0.84 a W). The RT-PCR assay developed appears to be a promising tool in the prediction of potential aflatoxigenic risk in stored peanuts, even in case of low-level infections, and suitable for rapid, automated and high throughput analysis.