Selection And Preliminary Application Of Deoxynivalenol Nucleic Acid Aptamer

Deoxynivalenol(DON)is one of the most common mycotoxins produced by Fusarium,which mainly contaminates corn,wheat and other grains and their products.DON not only causes acute toxicity,liver toxicity,immunotoxicity,etc.,to humans and animals,but even induces cancer.Timely and sensitive detection of DON to prevent it from being ingested by humans and animals is the main measure to prevent DON from causing harm to humans and animals.Nucleic acid aptamer is a single-stranded oligonucleotide sequence that can form a specific tertiary structure through self-winding and folding to bind to the target with high affinity and specificity.And it can be derived from the exponentially enriched ligand system evolution technology.In this study,the Capture-SELEX method based on a fixed library was used to screen DON aptamers,the sequencing results of the enriched library were analyzed and verified,and the obtained aptamers were used to explore the application methods,which laid a foundation for the establishment of a rapid detection method for DON.Capture-SELEX based on a fixed library was used in this study,with a biotin-labeled capture sequence complementary to the partial sequence of the initial random ss DNA library of 80 nucleotides in length,so that the library was fixed to the magnetic beads.The ss DNA sequence with high affinity to DON was dissociated from the magnetic beads and amplified by PCR.The single-stranded DNA was prepared by the long-short-strand method,and then recovered and purified for the next round.By monitoring the library retention rate and Q-PCR library abundance,the process was ended after 7 rounds of screening.After each round of secondary library amplification and purification,it was sent to high-throughput sequencing.It was sent to high-throughput sequencing after each round of secondary library amplification and purification.22 candidate aptamers were selected to affinity analysis test by Eva Green fluorescent dye method and graphene oxide method after homology analysis and secondary structure prediction.And it showed that two of the sequences had a higher probability of affinity for DON.The affinity constant was measured by the colloidal gold method,and the affinity constant of Sep.5 was 28.87 n M,and the correlation coefficient R~2 was 0.9904.The instrument was verified by circular dichroism,and then the specific analysis experiment was performed.It showed that it had a certain specificity to DON.I tried the ELOSA method with Sep.5 to improve the detection sensitivity of DON,but didn’t succeed.