Preparation Of Aptamers Against Saxitoxin And Its Antibody And Establishment Of Saxitoxin Detection

Saxitoxin (STX) is one of most toxic guanamines neurotoxin and it is one of theimportant virulence factors in the occurrence of harmful red tide. The STX comesmainly from secondary metabolites of certain hazardous plankton in seawater orfreshwater, and accumulate through the food chain in the shellfish, crabs and otherseafood. When eating the STX contamination seafood by human or animal, can causeparalysis poisoning and death. In recent years, With the increasing marine pollution,harmful algal blooms (HAB) and red tides have occurred frequently, resulting inseafood poisoning have occurred in the world. In order to protect the public health andsafety from toxic hazards, method for STX determination must be developed. Severalmethods have been reported for the determination of STX using a variety oftechniques, including mouse bioassay, sodium channel blocking assay, highperformance liquid chromatography (HPLC) and immunological methods. Althoughthese methods could be used for qualitative detecting purpose, many drawbacksassociated with them, such as the high variability of the results, low sensitivity, timeconsuming, high instrumentation costs and expensive certified reference standardsrequired for the tests. Especially, used of STX standard in detection process increasedtest costs and brought a security risk for experimental operator. Therefore, the searchfor an alternative to the STX standard has became the key to solve the above problems.In this study, we aimed at applying the SELEX strategy to select aptamers targetingSTX or STX antibody. The aptamers obtained in this paper is hopeful for developmentthe nontoxic detection method for regulatory monitoring of STX in seafood products.To obtain the ssDNA aptamer targeting STX, a random ssDNA library with81ntin length containing a random sequence of40nt which were flanked constant regionswas synthesized. Then, the fixed probe was design which is complementary to thefixed sequence of the ssDNA library, and it was chemical modified for connecting thesurface of the microplates. The ssDNA library was fixed on the microplate by fixedprobe and the aptamer of STX selection process was produced by the target inducedallosteric dissociation SELEX. After15rounds of repeated selection, the enriched ssDNA library was cloned and sequenced, and all of the sequences were carefullyidentified by analysis and ELISA. Finally, we successfully obtained three aptamers ofSTX.At the same time, high-affinity ssDNA aptamer-targeting F(ab′)2fragments of STXantibodies were selected from a random ssDNA library by the SELEX strategy. After16rounds of repeated selection, the enriched ssDNA library was cloned andsequenced, and all of the sequences were carefully identified by analysis and ELISA.The candidate aptamers in the above identification were selected for furthercharacterization by icELISA and the equilibrium filtration method. We successfullyobtained an aptamer that mimics STX in antibody binding and a substitute for STX inaptamer form has been developed.Based on target-induced dissociation of allosteric SELEX mode, we havedesigned an STX non-toxic ELISA method based on aptamer allosteric dissociation.According to the principle of STX and STX aptamer specific binding andconformational change, combined with microplate nucleic acid hybridization andbiotin-avidin-horseradish peroxidase chromogenic system to achieve non-toxicdetection of STX. And the conditions of this detection method are optimized,including the length of fixed probe, the aptamer concentration and salt concentrationin the washing buffer. A liner dose-response standard curve was prepared by plottinglog [STX] versus inhibiting rates. The regression equation of the standard curve wasy=-6.4916x+99.006，with the correlation coefficient R2=0.9959. The linear range andthe lower limit detection were3.92～1600ng/mL and3.92ng/mL.In addition, wecarried out linear regression analysis of the correspondence relationship between STXand STX antibody aptamer, the result shows a correlation between the concentrationof STX standard and the concentration of STX antibody aptamer, lay the foundationfor the establishment of STX nontoxic detection method.