Real time PCR: a good tool to estimate mycotoxin contamination in pig diets

Abstract

Cereals and soybean are the main components of pig diets. Unfortunately, feed materials are often contaminated with fungi and their metabolites, which pose a potential threat to human and animal health. Therefore, this study was undertaken to evaluate the effectiveness of cultural methods and quantitative PCR for detecting fungi and their metabolites in pig diets, and to determine which plant components are responsible for mycotoxin contamination of feed. The presence of mycotoxin-producing fungi of the genera Fusarium, Penicillium and Aspergillus and their metabolites was determined in pig diets with different inclusion levels of various cereals and transgenic soybean meal. Six farm-made complete diets containing locally produced feed materials and imported soybean meal were investigated. The presence of the following fungi in pig diets was determined by microscopic observations of fungal cultures and by qPCR: trichothecene-producing Fusarium spp. (Tri5 gene), Penicillium verrucosum (rRNA) and Aspergillus ochraceus (PKS gene). The concentrations of mycotoxins (ochratoxin A (OTA) and zearalenone (ZEA)), trichothecenes (deoxynivalenol (DON), 3-acetyl-deoxynivalenol and T-2 toxin (T-2)) were analysed by HPLC. The results of the qPCR analysis demonstrated that the presence of DNA of mycotoxin-producing fungi and mycotoxins in pig diets was correlated with the inclusion levels of transgenic soybean meal and various cereals. The above correlation was validated by an analysis of Spearman’s rank correlation between the content of transgenic soybean meal and various cereals vs mycotoxin concentrations and the amount of DNA of toxin-producing fungi in pig diets. A significant positive correlation was found between: the percentage content of soybeans vs the concentrations of DON (R=0.93), trichothecenes (R=0.76) and T-2 (R=0.64), the percentage content of barley vs the concentrations of DON (R=0.50) and T-2 (R=0.49), the percentage content of triticale vs OTA levels (R=0.47), the percentage content of oats vs ZEA levels (0.50). A correlation was also noted between the percentage content of soybeans and the amount of DNA of trichothecene-producing Fusarium spp. (R=0.96). The results of this study indicate that pig diets are significantly contaminated with toxin-producing fungi and their metabolites, and that the quantification of DNA of mycotoxin-producing fungi is a reliable indicator of mycotoxin contamination of feed. Our findings can contribute to reducing the costs of analyses that should be routinely performed to minimise the entry of mycotoxins into the food chain.