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

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.

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.

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.

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.