Screening Of DON Aptamer And Establishment Of Two Rapid Detection Methods

Food safety and food nutrition have gradually become two hot spots of human concern and the supervision of food safety is crucial.Avoiding the intake of mycotoxins in food is an important guarantee for improving human health and the quality of human life.DON is a fungus toxin with good water solubility,which is easily to be appeared in the water and food.Animals will be poisoned by eating feed containing DON.When people or animals ingest excessive amounts of DON,acute symptoms such as nausea,vomiting,diarrhea,abdominal pain,headache,dizziness,and fever may occur.In severe cases,they may be life-threatening.DON is also called vomiting toxin.Therefore,it is urgent to solve the problem of DON in food and water.According to European Commission Regulation No.574/2011,the limit of DON in regulating feed for farm animals is 5 mg/kg,and the limit of DON in cereals is 1.25 mg/kg.At present,the conventional methods for detecting DON are:TLC,ELISA,array sensors,FPIA,SERS,LC-MS/MS)analysis,electrochemical precipitation method.But the disadvantages of these methods are:the instrument is expensive,the instrument operation is cumbersome,the experiment takes a long time,and the cost is high.Therefore,there is an urgent need to develop a method for detecting DON that can complete the detection in a short time,with low cost and simple operation.This paper is mainly based on exponentially enriched ligand system evolution technology(SELEX),DON aptmers were obtained in vitro screening,and established two methods for rapid detection of DON(Complementary strand competition method,Fluorescence quenching method)were established.Firstly,DON was used as a target and Sepharose 6B immunoaffinity material was used as a medium.After 8 rounds of rigorous screening,aptamer capable of binding DON is obtained.Secondly,the aptamer was selected after cloning and sequencing by ELONA to prove their specificity,sensitivity and affinity.Thirdly,two quantitative detection methods was established based on complementary strand competition method and fluorescence quenching method and commercial kits and fluorescence quenching methods were used to spike the actual samples for recovery.Finally,Molecular simulation docking technology was used to study the interaction mode between specific aptamers and DON.After SELEX,cloning and sequencing,two aptamers(DON-A15 and DON-A16)were obtained.The secondary structure of these two nucleic acid aptamers were analyzed.The d G of DON-A15=-11.53 kal/mol and DON-A16=-10.93 kal/mol.Both aptamers have strong binding ability.Secondly,ELONA was used to evaluate and compare the specificity,affinity and sensitivity of these two aptamers.Among them,the better aptamer DON A16 which can be specifically bound was identified and used for subsequent research.The study was based on DON-A16 to establish a complementary chain competition method.DON-A16 com was used as a recognition element,DON-A16 was combined with DON-A16 com to form a competitive mode with the DON and monitor the change of OD450 nm.Under the conditions of experimental optimization,the detection limit of the sensor is 488 ng/m L,and it can be showed as a good linear relationship.The linear curve equation is,y=-0.0063x+0.5528,and the linear correlation technical coefficient is,R~2=0.9795.A biosensor was also established based on GO aptamers that adsorbed non-specific aptamers to the target.Using FAM-labeled DON-A16 as a recognition probe,under the optimal experimental conditions,the minimum detection limit of DON is 200 ng/m L,the linear curve equation,y=-70.553x+81.731,and the linear correlation coefficient,R~2=0.985.The detection time of GO fluorescence quenching method was 3 h.In order to verify whether the constructed fluorescence quenching biosensor can be reliably used for the detection of actual samples.Commercial DON-ELISA and fluorescence quenching method were used to study the recovery rate(between 84%-93%)in different samples(oat,wheat,corn).The results show that our method has less influence on the matrix in complex samples and better repeatability.It can be used for the detection of actual samples.Finally,from the molecular simulation docking simulation,the main binding site and the three-dimensional structure of DON-A16 and DON were found.It provides a solid foundation for the mechanism to analyze the mode between aptamer and target.