Screening And Identification Of T-2 Toxin Nucleic Acid Aptamer And Its Visual Detection Method

T-2 toxin（T-2）is a highly toxic trichothecene A compound.Investigations show that its detection rate is extremely high in grains and livestock and poultry feeds.After passing through the food chain,it will increase the safety hazards of human food,a serious threat to human and animal health.Therefore,the establishment of an efficient,convenient,fast and accurate detection technology is essential to control food safety.Traditional instrumental analysis and immunological analysis have complex steps,long time of batch sample detection,and low possibility of real-time detection on site.Colorimetric biosensing analysis has been extremely used in drug monitoring,food safety and environmental pollution in recent years due to its simple preparation,low cost,fast detection speed,and naked-eye analysis and judgment.Aptamer can specifically bind to the screening target and is called"artificial antibody".It is screened in vitro,easy to synthesize and modify,has strong affinity and good stability.Aptamer is one of the ideal molecular recognition elements in biosensor construction.However,there are few Apt screening sequences for T-2 at present,with low affinity and high cross-reaction rate,which limit its wide application in biosensing.This study used the exponential enrichment ligand system evolution technology to screen and identify T-2 toxin high-specificity and high-affinity nucleic acid aptamer from ssDNA library,and colorimetric detection technology was constructed based on the screening sequence.A method for rapid detection of T-2 toxin in animal food and feed,such as muscle,liver and milk,was established,which was easy to judge by naked eyes,and provided a new fast detection idea for food safety supervision.1 Establishment of a non-immobilized GO-SELEX screening method for T-2toxin nucleic acid aptamersIn this experiment,the screening and identification of T-2 toxin nucleic acid aptamer was completed by using non-immobilized graphene oxide-SELEX.Firstly,the ssDNA library of 80 bp length（40 bp random sequence in the middle and 20 bp fixed sequence at each end）was used for aptamer screening,based on the strong non-specific adsorption of GO and ssDNA,the principle of weak adsorption of the ssDNA complex that specifically binds to T-2 toxin,13 rounds of forward/reverse screening were performed,and the screening pressure was enhanced by reducing the input amount,increasing the reverse screening and shortening the incubation time to obtain stronger specificity series of nucleic acid aptamers.The initial library was pretreated by heat denaturation and incubated with the screening target under temperature control,and then GO was added to adsorb unbound ssDNA.After centrifugation,PCR amplification andλExonuclease hydrolysis were performed to obtain a secondary screening library.After the recovery rate was basically stable,the recovered products were analyzed.Single clone sequencing was performed,and 20 different ssDNA sequences were obtained;then sequence alignment analysis was performed using MEGA-X,and 4 sequences of Seq H3-3,Seq H5-2,Seq Q12-1 and Seq Q13-2 were selected.For affinity analysis,the dissociation constants were23.04±8.06 nmol/L,7.49±2.18 nmol/L,74.43±21.79 nmol/L,43.11±17.81 nmol/L;finally,according to the principle that the smaller the dissociation constant,the stronger the affinity,the specificity of H5-2 sequence was analyzed,and the results showed that the Apt sequence obtained by screening had good specificity,filled the T-2 toxin-specific aptamer library,and provided new ideas for the detection of T-2.2 Colorimetric detection of T-2 toxin based on aptamer-based gold nanoparticlesIn this experiment,the Seq H5-2 sequence obtained by the above screening was used to establish a colorimetric detection method for T-2 toxin by utilizing the LSPR characteristics of gold nanoparticles.First,the Probe G sequence,which was partially complementary to the sequence of the T-2 nucleic acid aptamer and was rich in G bases,formed a hybrid duplex under high temperature heating conditions.During detection,the toxin can specifically bind to Apt to release the Probe G sequence.The combination of hemin can form hemin/G-quadruplex DNAzyme with peroxidase-like activity,and then catalyze hydrogen peroxide oxidation,slow down the generation of gold nano-ions in the detection system,and form gold nano-ions in different states,the color changed from red to blue,which realizes the visual detection of T-2 toxin,and at the same time,the absorbance can be measured by the microplate reader to achieve accurate quantitative analysis.During the experiment,the concentration of MES,chloroauric acid,hydrogen peroxide,hemin,as well as the incubation temperature and incubation time were optimized in series,and the best detection conditions were obtained.In the range of 1μg/L to 100μg/L,the linear relationship between OD_550nmdetection value and T-2 toxin concentration was good,the regression equation y=-0.0005x+0.3846,R²=0.9908,and the detection sensitivity was 2.22μg/L,the visualize critical values was 20μg/L.Otherwise,the LOD for feed,pork,liver and milk samples were 119.80μg/kg,6.36μg/kg,7.27μg/kg,7.36μg/kg,respectively,and the limit of quantification（LOQ）were294.80μg/kg,15.81μg/kg,18.39μg/kg,18.77μg/kg.And after adding low,medium and high concentrations of T-2 toxin to the matrix,the recovery rates were all between66.67%and 96.25%,and the CV values within and between groups were all below 15%.After comparison,the colorimetric detection method had a good correlation with the LC-MS/MS method,and the correlation index was 0.9940,indicating that the detection and analysis method was reliable.In this study,based on the non-fixed GO-SELEX technology,the nucleic acid aptamer sequence Seq H5-2 of T-2 was screened and determined,and an Apt-based colorimetric method for T-2 toxin was established,which can be used in feed and animal-derived food.The rapid and simple detection of the product provides a new method for point-of-care detection in the fields of livestock and poultry breeding and food safety.