Selection,Identification And Application Of A DNA Aptamer Targeted To Ochratoxin A

Ochratoxin A(OTA)is a toxic secondary metabolite mainly produced by Aspergillus and Penicillium species,which widely exits in cereals and other food products.Ochratoxin A is a potent very harmful to humans and animals.Aptamers are single-stranded oligonucleotides(ssDNA or RNA)obtained by systematic evolution of ligands by exponential enrichment(SELEX)in vitro screening.They can bind target molecules with high affinity and specificity as a result of forming into a particularly complex spatial three-dimensional structures.In this work,aptamers for OTA have been obtained by a modified GO(graphene oxide)-SELEX methods.Then,two of fast,sensitive and visual analysis method for detecting OTA were constructed by combining the specific aptamer with GO quenching fluorescence and colloidal gold spectrophotometrc analysis techniques.Firstly,based on the principle that GO adsorbed to ssDNA,the OTA aptamers were screened using the immobilized GO-SELEX method.After 10 rounds of screening,the aptamer-enriched library was obtained and 23 aptamer candidate sequences were obtained after cloning and sequencing.Candidate sequences were selected for affinity and specificity analysis,after primary and secondary structure analysis.Finally obtained a best aptamer Seq.14 that specifically recognizes OTA toxins.The dissociation constant was 138.9±4.9 nmol/L.Secondly,a fluorescence sensor was constructed based on fluorescence resonance energy transfer(FRET)between carboxyfluorescein(FAM)labeled aptamer and GO.In the absence of OTA,the FAM-labeled aptamer is adsorbed and quenched by GO;In the presence of OTA,the aptamer binds to OTA and falls off the GO,causing a resumption of fluorescence.Under the optimized conditions,the OTA concentration was linearly correlated with fluorescence intensity between 20 and 300 nmol/L(R2=0.995),and the detection limit of the method was 21.8 nmol/L.The spiked recoveries were 96.0-119.7%,and the relative standard deviation was 3.8%.This method can apply to detect OTA toxin in red wine samples.Lastly,a simple and rapid method for the detection of ochratoxin A(OTA)was established based on the specificity of the aptamer for the target and the sensitivity of gold nanoparticles spectrophotometry.The aptamer can inhibit the aggregation of AuNPs in high salt solution.In the presence of OTA,it bound specifically with the aptamer,causing the AuNPs to aggregate and the color to change.Under optimal conditions,the concentration of OTA in the range of 0-300 ng/mL exhibited a good linear relationship with the ratio of absorbance(A656nm/A520nm).The linear regression equation was y=0.001x+0.2264,the coefficient of determination R2 was 0.9962,the spiked recoveries were 99.2?103.1%,and the relative standard deviation was 4.4%.