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. 2023 Sep;9(3):1-7.
doi: 10.22034/CMM.2023.345055.1420.

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

Amin Daliri  1 , Masoomeh Shams-Ghahfarokhi  1 , Mehdi Razzaghi-Abyaneh  2
Affiliations

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

Amin Daliri et al. Curr Med Mycol. 2023 Sep.

Abstract

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.

Keywords: Aflatoxin B1; Multiplex-PCR; Pistachio orchards; Aspergillus flavus.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Detection of Aspergillus flavus isolates producing aflatoxin B1 using the multiplex-polymerase chain reaction method. Lane 1: A. flavus 425, Lane 2: A. flavus 488, Lane 3: A. flavus 154, Lane 4: A. flavus 186, Lane 5: A. flavus 5004, Lane 6: Ladder (100 bp).
Figure 2
Figure 2
Dendrogram using multiplex polymerase chain reaction bands and the resultant classification between the binding patterns categories and toxin production.
See this image and copyright information in PMC

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