Screening And Preliminary Application Of A SsDNA Aptamer For Detection Of Brevetoxin-2

Brevetoxin-2is a kind of polyrther lipid solubility shellfish toxin which was isolated fromK.brevis.It is the most common neurotoxic shellfish poison (NSP) which comes from somespecies of poisonous algae occurred during the red tide and it can be calculated via the food web.Ingestion of contaminated molluscan shellfish may cause a cluster of gastrointestinal andneurolofical symptoms such as ataxia, slurred speech, nausea and vomiting, respiratory distressand partial limb paralysis. Recently, red tide occurred frequently because of the seriouslypollution of the environment. Bromatoxisms by eating contaminated shellfish by NSP arereported. This not only caused huge economic lossed to aquaculture and processing, but alsobrought a great threat to human health. In order to keep the public away from hidden dangers oftoxins and guarantee the development of the aquaculture industry, to strengthen the detection andsupervision of BTX-2become an effective way to aolve this problem. At present, the detection ofBTX-2mainly concludes mouse bioassay, high performance liquid chromatography, receptorbinding assay and radioimmunoassay and so on. These methods have their respective advantagesand disadvantages, the deficiency is need professional operation, expensive equipment, lowsensitivity, animal welfare.etc. In order to solve the problems above, to seek a new fast method todetect BTX-2accurately is of great significance.In rapid immunological detection method, theantibody can be chosen as detection probe, in addiction to aptamer, known as “chemicalantibody”. In order to obtain sensitive probe specific to BTX-2and develop rapid immunologicaldetection method, we selected aptamers with high affinity and specicifity to BTX-2usingSELEX technology. The selected aptamer was used as a detection probe to establish ELISAmethod.The research coupled the BTX-2with ovalbumin (OVA) and bovine serum albumin (BSA)using mixed anhydride method as the target. We designed and synthesized a random oligonucleotides library contained85nucleotides. Both ends of the sequence are respectivelycontaining22and23nucleotide fixed sequence to design synthetic primers, while in the middleis the random sequence containing40nucleotides. One of the primers was marked with biotinwhich was used for the amplification of secondary library and determination of affinity.Immobilized the target into microplate, screened aptamers with SELEX, implicated the libraryby PCR and identitied it via electrophoresis. After12rounds repeated screening and enrichment,the implificated secondary library with high affinity was linked, converted, cloned andsequenced. DNAMAN was used for the homology the analysis of primary structure andRNAstructure for the secondary structure prediction analysis of the sequences obtained above.The candidate aptamer’s affinity and specificity were identified by indirect ELISA. The screenedaptamer B5was used to establish indirect competitive ELISA, and the conditions were optimized.The standard curve equation of linear regression obtained was y=-33.219x+80.994, correlationcoefficient was R2=0.9875, the linear range of detection is from1.07ng/mL to68.57ng/mL andthe lowest detectable limit was0.835ng/mL. The establishment of competitive ELISA methodbased on aptamer detection probe provides alternative rapid detection method for developmentwith independent intellectual property rights in BTX detection in seafood. It also provideexperimental basis for the development of rapid detection kit and this research of BTX rapiddetection method has a vital significance to protect the safety of seafood consumption.