Rapid Detection and Identification of Mycotoxigenic Fungi and Mycotoxins in Stored Wheat Grain

Abstract

This study aimed to assess the occurrence of toxigenic fungi and mycotoxin contamination in stored wheat grains by using advanced molecular and analytical techniques. A multiplex polymerase chain reaction (PCR) strategy was established for rapid identification of mycotoxigenic fungi, and an improved analytical method was developed for simultaneous multi-mycotoxin determination in wheat grains by liquid chromatography-tandem mass spectrometry (LC/MS/MS) without the need for any clean-up. The optimized multiplex PCR method was highly specific in detecting fungal species containing species-specific and mycotoxin metabolic pathway genes. The method was applied for evaluation of 34 wheat grain samples collected from storage warehouses for the presence of mycotoxin-producing fungi, and a few samples were found positive for Fusarium and Aspergillus species. Further chemical analysis revealed that 17 samples contained mycotoxins above the level of detection, but only six samples were found to be contaminated over the EU regulatory limits with at least one mycotoxin. Aflatoxin B₁, fumonisins, and deoxynivalenol were the most common toxins found in these samples. The results showed a strong correlation between the presence of mycotoxin biosynthesis genes as analyzed by multiplex PCR and mycotoxin detection by LC/MS/MS. The present findings indicate that a combined approach might provide rapid, accurate, and sensitive detection of mycotoxigenic species and mycotoxins in wheat grains.

Keywords: LC/MS/MS; aflatoxins; deoxynivalenol; fumonisins; multiplex PCR; toxigenic fungi; wheat grains.

Figure 1

Specificity and sensitivity of multiplex PCR assays using species specific primers. (A) Specificity test. Lanes: M-100 bp DNA ladder; 1: primer set I; 2: primer set II; 3: primer set III; 4: primer set IV; 5: primer set V; and (B) sensitivity test. Lanes: 1: negative control; 2: positive control (wheat sample inoculated by A. parasiticus NRRL 6111, A. fumigatus NRRL 62427, A. carbonarius NRRL 368 and incubated for 48 hrs); 3–5 (represent samples inoculated by A. parasiticus NRRL 6111, A.fumigatus NRRL 62427, A. carbonarius NRRL 368 without incubation): 3: 10⁴ spores/g; 4: 10⁵ spores/g; and 5: 10⁶ spores/g.

Figure 2

Specificity of multiplex PCR assays using sets of primers for mycotoxin biosynthetic genes. (A) Primer set VI. Lanes: M: 1 kb DNA ladder; 1: A. parasiticus NRRL 6111; 2: A. flavus NRRL 3518; 3: A. flavus SS1; 4: A. flavus SS2; (B) primer set VII. Lanes: 1: F. verticillioides NRRL 25457; 2: F. sporotrichioides NRRL 3299; 3: F. culmorum NRRL 13320; 4: F. graminearum NRRL 3376; 5: F. verticillioides SS4; 6: F. verticillioides SS5; and (C) primer set VIII. Lanes: 1: P. verrucosum NRRL 965; 2: P. viridicatum NRRL 5571; 3: A. carbonarius NRRL 368; and 4: A. ochraceus NRRL 35018.

Figure 3

Specificity of multiplex PCR assays in artificially contaminated wheat grain samples. (A) Primer set VI. Lanes: M: 1 kb DNA ladder; 1: negative control (no inoculation); 2: wheat sample artificially inoculated with A. parasiticus NRRL 6111; and (B) primer set VII. Lanes: 1: negative control (no inoculation); 2: wheat sample artificially inoculated with Fusarium spp.

Figure 4

Multiplex PCR using DNA isolated from stored wheat grain. (A) Primer set VI. Lanes: 1: negative control; 2–6: wheat grain naturally contaminated by aflatoxicgenic Aspergillus spp.; and (B) primer set VII. Lanes: M: 100 bp DNA ladder; 1: negative control; 2–8: wheat grain naturally contaminated by toxicgenic Fusarium spp.