Aims: To develop a multiplex reverse transciption-polymerase chain reaction (RT-PCR) protocol to discriminate aflatoxin-producing from aflatoxin-nonproducing strains of Aspergillus flavus. Methods and Results: The protocol was first optimized on a set of strains obtained from laboratory collections and then validated on A. flavus strains isolated from corn grains collected in the fields of the Po Valley (Italy). Five genes of the aflatoxin gene cluster of A. flavus, two regulatory (aflR and aflS) and three structural (aflD, aflO and aflQ), were targeted with specific primers to highlight their expression in mycelia cultivated under inducing conditions for aflatoxins production. 48-h-old cultures expressed the complete set of the genes analysed here whereas 24-h-old ones did not. Genomic PCR (quadruplex PCR) was also performed in parallel using chromosomal DNA extracted from the same set of strains to correlate the integrity of the genes with their expression. Conclusions: We show that a good correlation exists between gene expression of the aflatoxin genes, here analysed by multipex RT-PCR, and aflatoxin production, except for one strain that apparently transcribed all the relevant genes but did not produce aflatoxin in the medium. Significance and Impact of the Study: This is the first example of the application of a combination of multiplex PCR and RT-PCR approaches to screen a population of A. flavus for the presence of aflatoxigenic and nonaflatoxigenic strains. The proposed protocol will be helpful in evaluating the risk posed by A. flavus in natural environments and might also be a useful tool to monitor its presence during the processing steps of food and feed commodities.
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