Abstract
Aspergillus flavus and A. parasiticus are two species able to produce aflatoxins in foodstuffs, and in particular in hazelnuts, at harvest and during postharvest phase. As not all the strains of these species are aflatoxin producers, it is necessary to develop techniques that can detect aflatoxigenic from not aflatoxigenic strains. Two assays, a LAMP (loop-mediated isothermal amplification) and a real time PCR with TaqMan® probe were designed and validated in terms of specificity, sensitivity, reproducibility, and repeatability. The capability of the strains to produce aflatoxins was measured in vitro and both assays showed to be specific for the aflatoxigenic strains of A. flavus and A. parasiticus. The limit of detection of the LAMP assay was 100–999 picograms of DNA, while the qPCR detected 160 femtograms of DNA in hazelnuts. Both techniques were validated using artificially inoculated hazelnuts and naturally infected hazelnuts. The qPCR was able to detect as few as eight cells of aflatoxigenic Aspergillus in naturally infected hazelnut. The combination of the LAMP assay, which can be performed in less than an hour, as screening method, with the high sensitivity of the qPCR, as confirmation assay, is able to detect aflatoxigenic strains already in field, helping to preserve the food safety of hazelnuts.
Highlights
Aflatoxins (AFs) are toxic polyketide secondary metabolites with low molecular weight and hepatocarcinogenic activity
We developed three different DNA-based methods, an end-point PCR, a loop-mediated isothermal amplification (LAMP) assay, and a qPCR assay, to detect the presence of two of the most common aflatoxigenic species belonging to Aspergillus section Flavi that are important contaminants in hazelnuts, A. flavus and A. parasiticus
Ninety strains of Aspergillus section Flavi isolated from hazelnuts (50 strains) (Table 1) and chestnuts
Summary
Aflatoxins (AFs) are toxic polyketide secondary metabolites with low molecular weight and hepatocarcinogenic activity. These compounds are poorly soluble in water and highly soluble in moderately polar solvents, such as chloroform, methanol, or dimethylsulfoxide. Regarding their chemical structure, AFs can be divided into two groups identified based on the fluorescence emitted under ultraviolet light: type B (blue fluorescence) with a difuro-coumaro-cyclopentenone structure; type G (green fluorescence) with a six-member lactone ring replacing the cyclopentenone. Eighteen different types of AFs are currently identified [1], with the most important and common aflatoxins in nature being aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1), and G2 (AFG2).
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