摘要
人工合成78个碱基的随机ss DNA文库,采用指数富集配基的系统进化技术与诱变聚合酶链式反应(polymerase chain reaction)结合的方法,通过筛选富集、克隆、测序,获得了与河豚毒素(tetrodotoxin,TTX)特异结合的单克隆DNA适配子A3。DNA适配子A3的二级结构主要为茎环结构,与TTX的亲和力为1.254。对适配子和TTX结合的磷酸盐缓冲液p H值、荧光染料结合时间进行优化,结果表明,最适p H值为7.5,最佳结合时间为10 min。建立的快速筛选检测TTX的DNA适配子-荧光染料法对TTX的检出限为10-6 mo1/L。
A 78-mer ss DNA library was synthesized in vitro. The tetrodotoxin (TTX) specific monoclonal DNA aptamer A3 was prepared using systematic evolution of ligands by exponential enrichment(SELEX) combined with mutagenic PCR by screening, enrichment, cloning and sequencing. The secondary structure of the DNA aptamer A3 mainly contained stem ring structure, and the affinity for tetrodotoxin was 1.254. The PBS buffer p H and fluorochrome-binding time were optimized. The results showed that the optimum p H was 7.5 and the best binding time was 10 min. As a result, a DNA aptamerfluorochrome method for rapidly screening and detecting tetrodotoxin was developed with a detection limit of 10-6 mo1/L.
出处
《食品科学》
EI
CAS
CSCD
北大核心
2014年第24期205-208,共4页
Food Science
基金
国家质检总局科研项目(2010IK145
2014IK104)
