Detection of Aflatoxin B1-producing Aspergillus flavus strains from pistachio orchards soil in Iran by multiplex polymerase chain reaction method.

Background and purpose: The current study aimed to report a multiplex polymerase chain reaction (PCR) assay as a monitoring technique to differentiate aflatoxigenic from non-aflatoxigenic strains of Aspergillus flavus isolated from pistachio orchards soil.

Materials and methods: In total, 25 A. flavus strains were isolated from soil samples of pistachio orchards. To test the strains for Aflatoxin B₁ (AFB₁)-producing ability, thin-layer chromatography (TLC) was used and the amounts of AFB₁ were measured by high-performance liquid chromatography (HPLC). Multiplex PCR was used as a genome-based method to detect genes responsible for AFB₁ production by A. flavus and the results were analyzed in terms of speed and specificity of detection. A set of four primers was designed specifically for the omtA, omtB, ver-1, and aflR genes which are commonly present in aflatoxin biosynthetic pathways.

Results: The AFB₁ production by the A. flavus strains ranged from 0 to 321 ρg/μl. Four-band patterns of the primer sets were observed only in AFB₁-producing A. flavus strains. Moreover, 18 out of the 25 strains showed all four bands belonging to omtA, omtB, ver-1, and aflR, whereas 7 strains did not display omtA, or aflR-related bands, in non-toxigenic and low toxin-producing A. flavus.

Conclusion: The multiplex PCR is a supplementary strategy to current conventional mycotoxin analytical techniques, such as TLC and HPLC. It could be used as an efficient method to differentiate aflatoxigenic from non-aflatoxigenic strains of A. flavus. This achievement is crucial to minimize fungal contamination of food, feed, and agricultural commodities, thereby reducing the risk of subsequent aflatoxin consumption.