The Method To Detect Three Mycotoxins In Corn

Mycotoxins result in reduced feeding value of corn, which leads to the negative effect on livestock growth performance and organ function, even the death of animals. Aflatoxin B1(AFB1), zearalenone (ZEN) and deoxynivalenol (DON) are three kinds of toxins which are investigated widely and whose pollution is the most common. Different doses of mycotoxins can cause different degrees of threat to the health of animals. Therefore, it is critical to determine the mycotoxins and toxin-producing molds. There are several detection methods available, such as thin-layer chromatography, liquid chromatography and immunochemical analysis. These methods are mainly based on the physical and chemical properties of mycotoxins and can not be applied to checking out the high-risk samples that may carry toxin-producing molds without detectable toxins. Multiple polymerase chain reactions can identify multiplex DNA templates or different areas of the same template at the same time in one single reaction lane. It is fast, efficient and cheap. This method is widely used in diagnosis of pathogens or microorganisms.The experiments were conducted to study the contamination of AFB1, ZEN and DON in corns from Shandong province in the autumn of2012and analyze the correlation of mycotoxins with moisture content, breakage rate and mildew rate of the corns. Aspergillus and Fusarium are two kings of fungi which can produce mycotoxins. In this study, we used their genome DNA as templates and designed several pairs of primers with several key genes involved in the biological synthesis of AFB1, ZEN and DON. We developed a multiplex PCR method which could rapidly and sensitively identify hidden toxin-producing fungi in the samples.1) We collected87corns from several typical feed enterprises, and measured the content of AFB1, ZEN and DON by ELISA. The detectable rate of AFB1, ZEN and DON were98.85%,100%and100%respectively. The exceeding rate beyond limitation standard of ZEN was the highest, which was12.64%. The exceeding rate beyond limitation standard of DON was the second higher, which was6.90%and AFB1’s was3.45%, which was the lowest. The linear correlation of ZEN and DON was significant with a P value of0.0008and a correlation coefficient of0.1368.2) We detected the moisture content following the procedure of Chinese standard of GB13078-2001, detected the breakage rate and mildew rate of every sample with statistical methods. The correlations of moisture with the contents of AFB1, ZEN and DON were analyzed. In all samples, the highest contents of free-water, bound water and moisture were14.06%,5.95%and19.24%respectively; the lowest contents of them were4.79%,1.11%and9.46%respectively, and the mean contents of them were9.44%,4.02%and13.46%respectively. The mean value of breakage rate and mildew rate were5.08%and6.08%. The highest mildew rate was27.20%and the highest breakage rate was31.80%. The linear correlation of mildew rate and ZEN content was highly significant, with a p value of0.0058. The linear correlation of mildew rate and breakage rate was significant, with a p value of0.0354.3) In this study, four pairs of primers were designed based on the sequence of aflR, PKS13and tri5, which were involved in the biosynthesis of AFB1, ZEN and DON and the sequence of β-Tubulin, which was involved in all fungi. These primers were used to establish a rapidly and sensitively multiplex PCR method to detect the hidden toxin-producing fungi in corn for feed production. With optimizing the reaction step by step, we developed a preliminary mPCR method. The method amplify the four target genes, which were400bp (aflR),658bp (tri5),537bp (P-Tubulin) and192bp(PKS13). The four target bands were clear with this optimized mPCR methods to amplify the fungal DNA extracted from corns.