The morphological descriptions of fungi have been increasingly replaced by the use of DNA molecular techniques for fungal identification at a species level. A simple, rapid and cost-effective protocol for efficiently extracting fungal genomic DNA for applications including DNA barcoding and genetic epidemiology is desirable. The objective of this work is to develop a rapid extraction method for ribosomal DNA (rDNA) directly from the axenic culture of fungi and yeasts that yield template pure enough for polymerase chain reaction (PCR) analysis of the internal transcribed spacer (ITS). A total of 254 fungal isolates were used to DNA extraction using our developed protocol. The PCR amplification of universal primers ITS1 and ITS4 shows the high sensitivity of DNA amplification with the right size for all tested species. This protocol is easy to perform and allowing processing a sample per minute, inexpensive; and it does not require the use of hazardous solvents.
Fungal contamination in nuts is unavoidable and is a major challenge to nuts safety, quality and then for human health after consumption particularly immunocompromised individuals. This study aimed to early detection of potential pathogenic Aspergillus in nuts to avoid the harmful effects of allergens and mycotoxins. Three different geographic locations in Basra, Iraq were screened, and a total of 28 fungal isolates were recovered from tested nuts including cashews, almonds, walnuts, pistachio and sunflower seeds. ELISA was used to identify isolated fungi based on antigenic structure via using a highly specific MAb for Aspergillus, JF5. The accuracy of ELISA was validated by ITS-rDNA sequence in comparison with NCBI database. The highest incidence of isolated fungi belongs to main four genera Aspergillus (78.57%), Alternaria (10.71%), Cladosporium (7.14%), followed by Penicillium (3.75%). The most dominant species was A. flavus, a potential aflatoxins producer, (28.57%). Abu was highly contaminated geographic site comparing with Zubayr and Qarmat. These findings raise awareness about contaminated nuts particularly there is no database available and highlighted the accuracy of using specific MAb for fungal identification and diagnosis. The occurrence of well-known toxigenic species such as A. niger, A. flavus and A. terreus suggested the possible risk of mycotoxin contamination of the nuts for human consumption.
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