基于分子信标的有机磷农药核酸适体活性鉴定

Activity identification of aptamers recognizing four organophosphorus pesticides based on molecular beacon

利用有机磷农药与分子信标竞争结合核酸适体的原理,建立了核酸适体活性的荧光鉴定方法,对前期筛选的15条核酸适体的活性进行了鉴定。结果表明,分子信标设计合理,甲醇和4种有机磷农药对分子信标发夹结构的稳定性影响较小,室温孵育时间为60 min时体系荧光强度趋于稳定,当体系中适体与分子信标的添加比例为1∶5时,荧光强度的净增量达到最大值。适体鉴定结果表明适体ss4,ss6,ss12,ss16,ss24和ss27对甲基对硫磷表现出了一定的活性,但亲和活性均较弱;适体ss5,ss12,ss13和ss24对马拉硫磷表现出了一定的活性,其中适体ss5活性最好,荧光抑制率达到了64.97%;适体ss1,ss4,ss6,ss7,ss10,ss16,ss17,ss23,ss27和ss29对辛硫磷表现出了一定的活性,其中适体ss27活性最好,荧光抑制率达到了49.42%;适体ss13,ss26和ss27对乐果表现出了一定的活性,其中ss26活性最好,荧光抑制率达到了44.35%。

The activities of the aptamers were identified using fluorescence assay based on organophosphorus pesticides and molecular beacon competitively binding to the aptamer in this paper. The results showed that the molecular beacon was rational, methanol and 4 organophosphorus pesticides less affected the stability of molecular beacon＇s hairpin structure. The fluorescence value in the system was stable when the incubation time was 60 min at room temperature. The net increment of fluorescence intensity value reached maximum when the ratio between aptamers and the molecular beacon reached to 1 ： 5. Identification results of aptamers showed that aptamers ss4, ss6, ss12, ss16, ss24, and ss27 were active to parathion-methyl, but their affinity activities were weak; aptamers ss5, ss12, ssl3 and ss24 were active to malathion, and ss5 had the best affinity, the fluorescence inhibition efficiency reached to 64.97%; aptamers ssl, ss4, ss6, ss7, sslO, ssl6, ssl7, ss23, ss27 and ss29 were active to phoxim, and ss26 had the best affinity, the fluorescence inhibition efficiency reached to 49.42% ; aptamers ssl3, ss26 and ss27 were active to phoxim, and ss27 had the best affinity, the fluorescence inhibition efficiency reached to 44.35 %.