Extraction of DNA to detection of aflatoxigenic Aspergillus species in foodstuffs using PCR method

Abstract

The extraction of DNA according to Cenis was used with different modifications of disrupting the cell wall. Fungi mycelium was disrupted by ultrasonic bath, Eppendorf homogenizer and liquid nitrogen. The method was optimized using pure cultures A. parasiticus var. globosus CCM F - 550 and A. flavus CCM F - 108. After extraction, acquired DNA was amplified by Polymerase Chain Reaction (PCR). PCR was used to amplify ver-1 gene and apa-2 gene as target fragments. The specific PCR product codes the biosynthetic way of aflatoxin B1. PCR product was detected by electrophoresis. Records were interpreted. Results acquired demonstrate, that success of method depends especially on disrupting cell wall of fungi. Altogether 91 samples of food (first of all tea, spices and medical herbs) were examined, and 27 samples were positive for the presence of the aflatoxinogenic fungi on the AFPA medium. Two samples contained two fungi species of genus Aspergillus and one sample contained three fungi species of genus Aspergillus. Finally, we determined 31 kinds of the potentially aflatoxinogenic fungi on the AFPA medium. The PCR were positive for 27 kinds of the aflatoxinogenic fungi.