Development and Optimization of a PCR–RFLP Method for Differentiation of Aspergillus flavus From Aspergillus parasiticus

Introduction: Aflatoxins are highly toxic secondary metabolites produced by several Aspergillus species, pose a global threat to food and feed safety, making rapid and reliable identification of aflatoxigenic species essential for effective surveillance and risk mitigation. Here, we report the development and validation a simple polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP) assay that discriminates between the closely related species Aspergillus flavus and Aspergillus parasiticus by targeting conserved regions of the aflatoxin biosynthetic pathway for use in feed surveillance. Materials & Methods: A total of ۴۲ fungal isolates from feed samples were identified as Aspergillus species and genomic DNA was extracted from the cultured mycelial biomass for further molecular analyses. The conserved primer sets to amplify three genes related to aflatoxin production pathway: aflD (nor-۱), aflR, and aflP (omtA). The PCR products were digested with the restriction enzymes XbaI and XhoI and resolved them using ۲% agarose gel. The RFLP patterns compared with the predicted digestion patterns from reference genomes to check their agreement and ability to provide accurate diagnosis. Results: Distinct RFLP patterns were obtained for A. flavus whereby for each of the three amplicons there were two diagnostic fragments (e.g. ۳۲۱/۱۳۹ bp for aflD). In contrast, the PCR products for A. parasiticus did not produce the same restriction sites, and no digestion was seen in this species. The assay consistently discriminated A. flavus from A. parasiticus.Conclusion: The recommended PCR–RFLP assay can be a rapid and cost-effective technique for identifying A. flavus from A. parasiticus in feed samples. However, using only this enzyme selection does not discriminate all clinically or agriculturally pertinent genera of Aspergillus. We recommend using this assay with added and new restriction enzymes, or coupling with other molecular analysis in order to allow for complete species discrimination. Ultimately, the performance should be validated with a variety of field isolates before using it as a standard diagnostic method.

کلیدواژه ها:

Aspergillus flavus ، Aspergillus parasiticus ، Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) ، aflD ، aflR ، AFLP