The Development And Application Of Detection Methods For Aflatoxigenic Aspergilli

Aflatoxins, as secondary metabolites of Aspergillus flavus and parasitic Aspergillus,were carcinogenic pollutions which are hard to eliminate from food industry. The detection ofaflatoxigenic Aspergilli before the toxin produced would be an effective way to controlaflatoxin contamination. Traditional media detection methods are so tedious, time-consumingand poor sensitive that demands of rapid, sensitive, real-time detection can’t be met. Study ofthe rapid method development is in urgent need.In this research, electrodes based on aflatoxin gene and specific antibody were preparedand three rapid detection methods based on PCR and immunological technique to detect nor-1gene and aflatoxigenic Aspergillus flavus and Aspergillus parasiticus were established. Andthen the established methods were applied to detect aflatoxigenic Aspergilli in soybean paste.Fluorescent quantitative PCR analysis system to detect aflatoxin biosynthetic nor-1genewas developed. This method was time consuming and sensitive. The whole detectionprocedure took only6hours and it was10times more sensitive than traditional methods.What’s more, the repetitive amplification variation factors of interclass were less than5%,which demonstrate that the method was stable.Using methylene blue as electrical indicator, DNA sensor aimed at nor-1gene ofaflatoxigenic Aspergilli was successfully designed based on graphene, CdTe-CdS quantum dot,gold nanoparticle and hybrid principle.The detection range of PCR products was0.0625-4ng/μL and the sensitivity to thesynthesized complement DNA was0.3×10-12mol/L(S/N=3), which provided possibility fordetection of trace amounts of nor-1gene of aflatoxigenic Aspergilli. Meanwhile, the sensorpossessed advantages such as label-free, easy to operate and rapid response. Theminiaturization of electrode is potential to realize real time detection of aflatoxigenicAspergilli.The quantitative PCR and DNA sensor could specifically aim at nor-1gene of Aspergilli.However, Owing to complexity and variety of genes involved to the toxins,the results showedthat quantitative PCR system also react to some strain of Aspergillus oryzae containing nor-1gene but without expression.In this study, highly specific immunological technique and sensitive sensor technologywas combined to make more specific immunosensors based on incorporation of L-Cys and AuNanoparticles (NPs). Injecting of the New Zealand white rabbit, serum antibody with25600potency unit was prepared.The specificity of antibody was excellent. The crossed reactionrates with the aflatoxigenic Aspergillus flavus and Aspergillus parasiticus were0.77-1.16andthey were just0.01-0.11with other14non-aflatoxigenic Aspergilli. The reaction ratesbetween the sensor and aflatoxigenic Aspergillus flavus and Aspergillus parasiticus couldreach87.89%-98.19%, while the reaction rates were lower than13%with othernon-aflatoxigenic strains.The sensor was fast, senstive and could be applied to detect realsamples. The detection range was75-1500spores/mL and its LOD was18spores/mL.The quantitative PCR and immunosensor were applied to the detection of nor-1gene and aflatoxigenic Aspergillus flavus and Aspergillus parasiticus successfully. Dynamic monitoringof total bacterial count, contaminated molds, AFB1, nor-1gene and aflatoxigenic Aspergillusflavus, Aspergillus parasiticus during the natural fermentation of soybean paste showed that53.85%of AFB1in fermentative soybean paste came from the raw material, while46.15%ofthat came from the productive process. The total bacterial count and aflatoxigenic Aspergillipollution may reach the maximum value in starter propagation stage, so starter propagationstage was the key stage for safe production of soybean paste.