Detection of Toxic Aspergillus Species in Food by a Multiplex PCR
Aspergillus parasiticus and flavus are known as the main producers carcinogen aflatoxins. The presence of these fungus and aflatoxins in food is a serious risk for human and animal health. The identification of these fungi is not straightforward due to its similarities with closely related species. In this study, a multiplex PCR assay have been developed which overcame the disadvantages in detection of Aspergillus parasiticus and flavus by conventional morphological method. Twelve isolates of filamentous fungi, representing six Aspergillus and Aspergillus-related species were used. DNA was extracted from mycelium following SDS method modified from Plaza’s method. The extracted DNA was used for the PCR method to identify the presence of selected fungi using specific primer sets. The result of identification was analysed by agarose gel electrophoresis. The sensitivity and specificity of the detection were checked by result of a gradient PCR assays. Two specific set of primers were successful designed based on Aflatoxin biosynthesis gene cluster to detect A. parasiticus and A. flavus species, with the limitation of detection (LOD) for each set of primer is 0.005ng and 0.008ng. A multiplex PCR assay set up with two these sets of primers was also successful in detection of both targets of A. parasiticus and A. flavus at the annealing temperature of 65°C with high sensitive and specificity. The limitation of detection (LOD) is 0.5ng. A highly specific multiplex PCR assay have been developed to detect the presence of Aspergillus parasiticus and flavus in food. This will permit prediction of the presence of aflatoxins type G and aflatoxins type B, the most potent natural carcinogens.
KeywordsAspergillusflavus Aspergillusparasiticus aflatoxin aflatoxin biosynthesis gene clusters norB − cypA genes
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