*3.3. Aspergillus flavus PCR Detection*

A total of 21 *Aspergillus flavus*, and *Aspergillus* related isolates such as two isolates for both *Aspergillus clavatus*, *Aspergillus ochraceous*, *Aspergillus niveus*, *Aspergillus terreus*, *Aspergillus fumigatus*, *Aspergillus versicolor*, *Penicillium paneum*, *Penicillium expansum*, *Penicillium citrinum*, *Penicillium verrucosum* in addition to one isolate from *Alternaria alternata* were used for testing specificity of primers. Figure 7 indicates the findings of the PCR product inspection of the agarose gel assay; the presence of a band at 236 bp for primers *aflP* and 412 bp *aflA* primers, respectively and suggested the same predicted PCR product. The samples were prepared independently from individual strains, with an identical amount of DNA (60 ng/µL). Figure 7 shows the results for screening the specificity of the tested primers. Thus, two sets of primer pairs (*aflP* and *aflA*) are specific for the detection of *A. flavus* (Figure 7A,C). The results show that only *A. flavus* DNA can be amplified (lines 1 to 21), no PCR products from other *Aspergillus* species and Penicillium were obtained from lane 1 to 21 (Figure 7B). For rapid and accurate detection of *A. flavus* isolates tested in the current report, two PCR primer sets (*aflP* and *aflA*) were employed. The applicable primers amplified a PCR fragment sited near the 18SrRNA region with 95.3% efficiency and 100% specificity. Ultimately, existing examinations suggest the powerful specificity of the *aflA* PCR primer over different typically available diagnostic primers for correct, speedy, and large-scale identification of *A. flavus* isolated from feeds. Most *A. flavus* strains were detected with the aid of specific-PCR by using the examined primer sets.

**Figure 7.** PCR amplicons obtained using primer pairs developed for the *aflP* (*omtA*) and *aflA* genes in 21 *Aspergillus flavus* (Lane1–21) tested with *aflP* primers (**A**), and an *Aspergillus* related isolate such as two isolates for both of *Aspergillus clavatus, Aspergillus ochraceous, Aspergillus niveus, Aspergillus terreus, Aspergillus fumigatus, Aspergillus versicolor, Penicillium paneum*, *Penicillium expansum*, *Penicillium citrinum, Penicillium verrucosum* and one *Alternaria alternate* isolate (Lane 1–21) tested with *aflP* primers (**B**). A total of 21 *Aspergillus flavus* (Lane1–21) tested with *aflA* primers (**C**).
