Research On Quantitative Detection Of Mycotoxins In Milk And Development Of Test Strips Based On Fluorescence Immunochromatography

Aflatoxin M₁(AFM₁)and Ochratoxin A(OTA)are common mycotoxins in milk.The constant accumulation in the body could damage the liver and kidney function and seriously endanger the health of humans and animals,the potential risk of AFM₁ and OTA coexisting in the human body and producing a combined toxic effect also increases.The current rapid detection methods for mycotoxins on the market are mainly complex enzyme-linked immunoassay(ELISA)and low sensitivity colloidal gold immunochromatography(CGICA),and most of them only target a single toxin,while multi-component rapid detection technology The research is only based on colloidal gold immunochromatography technology,it is difficult to achieve simultaneous and rapid quantitative detection of mycotoxins mixed pollution.Therefore,the establishment of a rapid detection technology for mixed mycotoxins is of great significance and application value for ensuring the safety of dairy products and the health of consumers.This topic is mainly based on the principle of immunochromatography,taking AFM₁ and OTA as the main research objects,using fluorescent microspheres as tracers to realize the single detection of AFM₁ and the dual rapid detection of AFM₁-OTA,which improved the detection efficiency and sensitivity of mycotoxins,it also provided a new idea for the simultaneous quantitative detection of mycotoxins mixed contamination in milk.The main research contents and results are as follows:(1)Based on the principle of immunological competition,AFM₁ fluorescence quantitative test strips were constructed.Spray the complete antigen and goat anti-mouse Ig G antibody on the nitrocellulose membrane respectively,prepare and characterize the fluorescent probe,the AFM₁ time-resolved fluorescence immunochromatographic quantitative detection method was established and applied to the detection of AFM₁ in dairy products.In the linear range of 0.05 ng/m L?2 ng/m L,the linear fitting equation of AFM₁ was Y=-45.53Lg X+26.68(R²=0.9870),the half maximal inhibitory concentration(IC₅₀)was0.2041 ng/m L,and the limit of detection(LOD)was 0.0194 ng/m L.The recovery rates of spiked milk samples were between 83.48%and 119.68%,and the relative standard deviations were all lower than 8.11%.Compared with the methods for detecting AFM₁ reported in recent years,this method has low detection limit and low cost,and provides a technical means for rapid screening and quantitative analysis.(2)In order to ensure stable and reliable quantitative signal output,an independent biotin-streptavidin(biotin-SA)system was introduced,and a dual-color fluorescence immunochromatography technique for simultaneous detection of AFM₁ and OTA was established to realize the simultaneous detection of mixed mycotoxins in milk.Different from the traditional method,this study used the target antigen and biotin-BSA as the detection line and the control line respectively,under the conditions of a pre-incubation time of 10 min,immunochromatography time of 12 min,a dual-color fluorescent probe dosage of 3?L,and a complete antigen spray concentration of 0.4 mg/m L,the linear fitting equation of AFM₁ was Y=-28.47Lg X+29.81(R²=0.9845),IC₅₀=0.2087 ng/m L,LOD=0.0527 ng/m L;the linear fitting equation of OTA was Y=-36.06Lg X+51.34(R²=0.9736),IC₅₀=1.1187 ng/m L,LOD=0.1662 ng/m L.The recovery rate of the traditional multiplex detection method was42.97%?140.80%,large fluctuation range,poor stability,the recovery rate was between90.16%?115.65%,and the coefficient of variation of this method was less than 7.41%.It could be stored continuously for 16 days at 37?,that is,the shelf life could reach more than one year under low temperature conditions.The method was applied to detect 14commercially available milk samples,and the results were basically consistent with the results of UPLC-MS/MS.(3)In order to further improve the detection sensitivity and stability of dual test strips,on the basis of traditional methods,an AFM₁-OTA dual time-resolved immunochromatographic detection technology based on the secondary antibody labeling method was established.By optimizing the labeled amount of antibodies,and the methanol concentration in the sample diluent,the detection sensitivity of this method was 0.0181 ng/m L and 0.0363 ng/m L,compared with the monoclonal antibody labeling method,it has been improved by 5 times and 3 times,the recovery rate of different batches was between 89.65%?103.99%.In the linear range of 0.05 ng/m L?5 ng/m L,the linear fitting equation of AFM₁ was Y=-27.71Lg X+28.70(R²=0.9901),IC₅₀=0.1696 ng/m L;In the linear range of 0.05 ng/m L?10ng/m L,the linear fitting equation of OTA was Y=-25.85Lg X+39.96(R²=0.9903),IC₅₀=0.4084 ng/m L,the detection linear range could reach two orders of magnitude.The interference experiment proved that the method has good specificity.The detection of different batches of different types of milk samples and further confirmation by the national standard method(R²>0.9789)showd that the technology is highly sensitive,accurate and reliable.It not only saved the amount of monoclonal antibodies,but also realized rapid quantitative detection in 15 min,which laid a good foundation for the simultaneous preliminary screening of large quantities of samples.