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. 2023 May 24;14(3):227-238.
doi: 10.1080/21501203.2023.2213704. eCollection 2023.

Development of a real-time PCR and multiplex PCR assay for the detection and identification of mycotoxigenic fungi in stored maize grains

Mayasar I Al-Zaban  1 , Ahlam H Alrokban  1 , Mohamed A Mahmoud  2
Affiliations

Development of a real-time PCR and multiplex PCR assay for the detection and identification of mycotoxigenic fungi in stored maize grains

Mayasar I Al-Zaban et al. Mycology. .

Abstract

This study aimed to identify important mycotoxigenic fungi and accurate detection of mycotoxin in stored maize grains using molecular methods. The current study also optimised the real-time PCR (RT-PCR) assay. The melting curve was established to identify isolated fungal species of Aspergillus (4), Fusarium (3), Penicillium (3), and Alternaria (one). A multiplex polymerase chain reaction (mPCR) technique was developed for the detection and characterisation of mycotoxin producing fungi, mycotoxin metabolic pathway genes, and the determination of eleven mycotoxins in stored maize grains using high-performance liquid chromatography (HPLC). The mPCR results indicated positive signals for potentially mycotoxigenic fungal species tested of Aspergillus, Fusarium, Penicillium, and Alternaria. A protocol for multiplex reverse transcription-polymerase chain reaction (mRT-PCR) was tested to distinguish between free and contaminated, stored maize with aflatoxin B1 (AFB1). The expression pattern of four aflatoxin biosynthetic pathway genes, AFB1 (aflQ, aflP, aflO, and aflD), was a good marker for contaminated, stored maize grains. HPLC analysis showed that maize grain samples were contaminated with mycotoxins, and the concentration was above the detection level. The results indicate that the polyphasic approach might provide a sensitive, rapid, and accurate method for detecting and identifying mycotoxigenic fungal species and mycotoxins in stored maize grains.

Keywords: HPLC; Real-time PCR; aflatoxins; fumonisins; maize grains; multiplex PCR; mycotoxigenic fungi; mycotoxins.

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Conflict of interest statement

No potential conflict of interest was reported by the author(s).

Figures

Figure 1.
Figure 1.
Multiplex PCR using mycotoxigenic fungal DNA isolated from stored maize grain naturally contaminated, Lanes: 1: Negative control; 2: Primer set I (Aspergillus spp.); 3: Primer set II (Fusarium spp.); 4: Primer set III (Penicillium spp.); 5: Primer set IV (Alternaria spp.).
Figure 2.
Figure 2.
The percentage of occurrence percentage, relative density of mycotoxigenic fungi, and contamination level in maize sample.
Figure 3.
Figure 3.
mPCR of stored maize grain. (a) Lane: M: 100 bp DNA ladder; Lanes: 1–2: Maize grain naturally contaminated by aflatoxigenic A. flavus, (using RNA, positive sample for four genes aflD, aflO, aflP, and aflQ), 3: Non-toxigenic isolates of A. flavus; 4: Non-toxigenic isolates of A. Niger;5: Non-toxigenic isolates of A. ochraceus, and (b) Lane 1: Maize grain naturally contaminated by toxigenic F. verticillioides (using DNA, positive sample for two genes fum6, fum8); 2: Non-toxigenic F. verticillioides; 3–4: Non-toxigenic F. proliferatum; 5: Non-toxigenic F. oxysporum.
See this image and copyright information in PMC

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