Detection and isolation of aflatoxin producing Aspergillus sp. in chewing and smokeless tobacco by microbial and molecular methods

Tobacco contains many harmful chemicals, toxins, and carcinogens that cause serious health hazards. Mycotoxins and aflatoxins are present in all forms of tobacco, including chewing tobacco and smokeless tobacco. Using microbial and molecular techniques, we identified and isolated the aflatoxins-producing fungus Aspergillus species. 50 samples were collected randomly and grown on SDA media by spread plate method. Aspergillus flavus isolates were tested for aflatoxin production using ammonia vapour test. Additionally, their DNA was extracted and utilized for molecular identification. Different fungal genera were observed, including the most commonly found Aspergillus as well as Penicillium, Rhizopus, Candida and Trichophyton. The isolated Aspergillus flavus was characterized by its soft, velvety white surface. The colony diameter ranged from 50 to 70 mm. When exposed to UV light at 365 nm on coconut agar medium, blue-green fluorescence was observed on the reverse side of the colonies. Further, the DNA extracted from all aflatoxin-producing strains was quantified. The study revealed high levels of aflatoxin in 10 different samples, while the remaining samples showed a lower range. The research employed both quantitative and qualitative techniques for the detection of aflatoxin-producing fungi. Notably, aflR and aflS were identified as potential candidates for the detection of aflatoxigenic fungi.