*4.4. Specific PCR Detection Method*

DNA from food samples was extracted using a QiaGene DNA extraction kit (Qia-Gene, Berlin, Germany). DNA was dissolved in DEPC-treated water, quantified spectrophotometrically and analyzed using 1.2% agarose gels. The AflR gene (744 bp) was amplified using specific primers. The PCR reaction consisted of 1 μL of DNA in 2.5 μL Taq polymerase buffer 10× (Promega, New York, NY, USA) containing a final concentration of 1 mM MgCl2, 0.2 Mm dNTPs, 20 pmol of each primer, and 0.2 μL Taq polymerase (5 U/μL) in a final reaction volume of 25 μL. The PCR reaction program was: initial denaturation at 95 ◦C for two minutes followed by 35 cycles of 58 ◦C for one min, 72 ◦C for one min, and 95 ◦C for 2 min. A final extension step at 72 ◦C for 5 min was included at the end of the reactions. PCR amplification products were separated in 1.5% agarose with 0.5× TBE buffer and visually analyzed with a gel documentation system (Syngene) [28]. Forward (5'-TAAGCAGAATTCGAATAGCTTCGCAGGGTGGT-'3) and reverse (5'-GAATAGCTTCGCAGGGTGGTGCGGCCGCTAAGCA-'3) primers were designed by Primer-Blast, NCBI.
