Identification of aspergillus parasiticus using PCR method

Abstract

Aspergillus parasiticus is one of the main producers of well known carcinogen aflatoxins. The presence of this fungus and aflatoxins in food is a serious risk for human and animal health. The identification of A. parasiticus is not straightforward due to similarities with closely related species, particularly A. flavus – an aflatoxin producing species. In this study, a PCR based method have been developed which overcame the disadvantages in detection of A. parasiticus by conventional morphological method. This method would provide a tool for accuracy, specificity and sensitivity in order to detect this species by using a couple specific primers designed on norB-cypA genes. A DNA isolation protocol with SDS, sand and thermal shock was also selected providing DNA solution for the detection of A. parasiticus by PCR test. Primers were optimized for best amplification results at temperature 62˚C. Discrimination capacity for detection of A. parasiticus of the test was examined by using samples with pure target DNA and with DNA mixture containing other related species at same ratios. The sensitivity of the test was identified at 0.005 ng/µl of target DNA. The method would be considered as valuable tool to improve diagnosis of aflatoxin at an early stage and in all critical control points of food chain integrated in HACCP strategies.

Key words: Aspergillus parasiticus, aflatoxins, aflatoxin gene cluster, PCR.