Molecular characterization of ochratoxigenic fungi associated with poultry feedstuffs in Saudi Arabia

Fungal and mycotoxins contamination of food and poultry feeds can occur at each step along the chain from grain production, storage, and processing. A total of 200 samples comprising of mixed poultry feedstuffs (n = 100) and their ingredients (n = 100) were collected from Riyadh, Alhassa, Qassium, and Jeddah cities in Saudi Arabia. These samples were screened for contamination by fungi. Penicillium chrysogenum was the predominant species taking into its account and frequency, respectively, in both mixed poultry feedstuff and barley samples (4,561.9 and 687 fungal colony-forming units (CFU)/g) and (66% and 17%). Moisture content was an important indicator for the count of fungi and ochratoxin A. Ochratoxin analysis of plate cultures was performed by a HPLC technique. Sample of mixed poultry feedstuff which was collected from Jeddah displayed the highest level of ochratoxin (14.8 µg/kg) and moisture content (11.5%). Corn grains samples were highly contaminated by ochratoxin A (450 and 423 µg/kg) and recorded the highest moisture contents (14.1 and 14.5%). Ochratoxin A production in fungal species isolated from mixed poultry feedstuff samples were high with P. verrucosum (5.5 μg/kg) and A. niger (1.1 μg/kg). In sorghum and corn grains, the highest ochratoxins producing species were P. viridicatum (5.9 μg/kg) and A. niger (1.3 μg/kg), respectively. Sixty-three isolates of A. niger were ochratoxigenic, and all of them showed the presence of pks genes using PKS15C-MeT and PKS15KS primer pairs. The detection technique of A. niger in poultry feedstuff samples described in the present study was successfully used as a rapid and specific protocol for early detection of A. niger without cultivation on specific media

Development of a Nanobody–Alkaline Phosphatase Fusion Protein and Its Application in a Highly Sensitive Direct Competitive Fluorescence Enzyme Immunoassay for Detection of Ochratoxin A in Cereal

A rapid and sensitive direct competitive fluorescence enzyme immunoassay (dc-FEIA) for ochratoxin A (OTA) based on a nanobody (Nb)–alkaline phosphatase (AP) fusion protein was developed. The VHH (variable domain of heavy chain antibody) gene of Nb28 was subcloned into the expression vector pecan45 containing the AP double-mutant gene. The Nb28–AP construct was transformed into Escherichia coli BL21­(DE3)­plysS, and soluble expression in bacteria was confirmed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot. Both the Nb properties and AP enzymatic activity were validated by colorimetric and fluorometric analysis. The 50% inhibitory concentration and the detection limit of the dc-FEIA were 0.13 and 0.04 ng/mL, respectively, with a linear range of 0.06–0.43 ng/mL. This assay was compared with LC–MS/MS, and the results indicated the reliability of Nb–AP fusion protein-based dc-FEIA for monitoring OTA contamination in cereal