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.

A simple and rapid method for the determination of ochratoxin A (OA) in ham was developed using a basic methanolic extraction, immunoaffinity column clean-up and a fluorometric determination of the toxin contamination levels. A mean recovery of OA from ham samples spiked at levels from 0.7 to 9.7 microg kg(-1) was 83 +/- 6% using the fluorometric method, with a detection limit of 0.7 microg kg(-1). Recovery data were compared statistically with those obtained using reversed-phase high-performance liquid chromatography with acetonitrile-water-acetic acid (99:99:2) as mobile phase and fluorescence detection, commonly used for OA determination in food. A good correlation between the two analytical techniques was obtained. Both methods were successfully applied to 42 ham samples, 21 in the middle of the ripening period (after 6 months from the process beginning) and the other 21 at the end of the maturation, after 12 months. Twenty-seven samples (64%) showed an OA contamination level <1.0 microg kg(-1), the Italian Ministry of Health guideline. The maximum contamination level found was 2.3 microg kg(-1). A good agreement (R(2) = 0.980) between HPLC and fluorometer analysis on naturally contaminated samples was obtained.

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An improved analytical method for aflatoxin B1 (AFB1) and aflatoxin M1 (AFM1) determination in pig liver is described, using an aqueous methanol extraction, an immunoaffinity column clean-up step and a direct fluorometric measurement for toxin detection and quantification. A detection limit of 1.0 microg kg-1 was achieved for AFB1 and AFM1. Mean recoveries of 80.7+/-9.0% for AFB1 spiked at 1.0-9.7 microg kg-1 levels and of 76.7+/-6.6% for AFM1 spiked at 1.0-5.5 microg kg-1 levels were obtained. Recovery data for spiked samples were statistically compared with those obtained by the same extract using classical reversed-phase high-performance liquid chromatography (RP-HPLC) with fluorescence detection, showing a significant correlation (p

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