In vitro Selection of DNAAptamers and Fluorescence-Based Recognition for Rapid Detection Listeria monocytogenes

Aptamers are specific nucleic acid sequences that can bind to a wide range of nucleic acid and non-nucleic acid targets with high affinity and specificity. Nucleic acid aptamers are selected in vitro from single stranded DNA or RNA ligands containing random sequences of up to a few hundred nucleotides. Systematic evolution of ligands by exponential enrichment （SELEX） was used to select and PCR amplify DNA sequences （aptamers） capable of binding to and detecting Listeria monocytogenes, one of the major food-borne pathogens. A simplified affinity separation approach was employed, in which L. monocytogenes in exponential （log） phase of growth was used as the separation target. A fluorescently-labeled aptamer assay scheme was devised for detecting L. monoeytogenes. This report described a novel approach to the detection of L. monocytogenes using DNA aptamers. Aptamers were developed by nine rounds of SELEX. A high affinity aptamer was successfully selected from the initial random DNA pool, and its secondary structure was also investigated. One of aptamers named e01 with the highest affinity was further tested in aptamer-peroxidase and aptamer-fluorescence staining protocols. This study has proved the principle that the whole-cell SELEX could be a promising technique to design aptamer-based molecular probes for dectection of pathogenic microorganisms without tedious isolation and purification of complex markers or targets.     

In vitro Selection of DNA Aptamers and Fluorescence-Based Recognition for Rapid Detection Listeria monocytogenes

Aptamers are specific nucleic acid sequences that can bind to a wide range of nucleic acid and non-nucleic acid targets with high affinity and specificity. Nucleic acid aptamers are selected in vitro from single stranded DNA or RNA ligands containing random sequences of up to a few hundred nucleotides. Systematic evolution of ligands by exponential enrichment (SELEX) was used to select and PCR amplify DNA sequences (aptamers) capable of binding to and detecting Listeria monocytogenes, one of the major food-borne pathogens. A simplified affinity separation approach was employed, in which L. monocytogenes in exponential (log) phase of growth was used as the separation target. A fluorescently-labeled aptamer assay scheme was devised for detecting L. monocytogenes. This report described a novel approach to the detection of L. monocytogenes using DNA aptamers. Aptamers were developed by nine rounds of SELEX. A high affinity aptamer was successfully selected from the initial random DNA pool, and its secondary structure was also investigated. One of aptamers named e01 with the highest affinity was further tested in aptamer-peroxidase and aptamer-fluorescence staining protocols. This study has proved the principle that the whole-cell SELEX could be a promising technique to design aptamer-based molecular probes for dectection of pathogenic microorganisms without tedious isolation and purification of complex markers or targets.     

核酸适体及其在疫病诊断上的应用

核酸适体是指采用指数富集配体的系统进化技术从随机单链寡核苷酸库中筛选出的能与靶物质高特异性、高亲和力结合的配体。鉴于适体特有的亲和力高、特异性强、精确识别、易体外合成与修饰等特点,其被广泛应用于分析化学、基因调控、蛋白质组学和新药研发等领域。在疫病的检测方面,核酸适体逐渐成为抗体的代替或补充试剂,已初步应用于生物芯片、生物传感器、分子信标等多种检测技术平台,并具有良好的敏感性和特异性,显示出了良好的应用前景。本文就适体技术及其在疫病诊断中的应用进展作一综述。     

基于纳米金和核酸适配体的重金属离子传感器研究进展

综述了国内外基于纳米金和核酸适配体的重金属离子传感器的研究进展,从比色法、荧光法和共振散射光谱法三大类详细阐述了各类传感器的原理、技术应用及优缺点,讨论了现有传感器存在的问题和未来检测技术的发展方向。     

适配体的研究进程与思考*

 适配体（aptamer）是指利用指数富集的配体系统进化（SELEX）技术，从人工合成的寡核苷酸文库中筛选获得的能够与靶分子特异结合的短单链DNA和RNA分子。筛选得到的适配体可以与DNA，RNA，蛋白质或其它靶分子结合，影响这些靶分子的性质，从而起到改变与靶分子相关的生物学功能的效果。而且研究发现，适配体在与靶分子相互作用时，不仅序列，它们所形成的三维结构也尤其重要。同时，适配体在生物医药研究方面显示出广阔的应用前景。介绍了适配体的发展历程，以及一些典型适配体的生物学功效。提出了RNA适配体三维结构在与靶分子进行相互作用时可能发挥重要作用，以期为RNA适配体的筛选提供理论思考。     

2种不同方法制备玉米赤霉烯酮单克隆抗体适配子的ssDNA文库的比较

体外合成全长为71个碱基的随机ssDNA文库,采用SELEX技术,以玉米赤霉烯酮单克隆抗体为靶蛋白,微孔板为筛选介质进行筛选。通过对比不对称PCR法和生物素-链霉亲和素磁珠2种分离方法制备ssDNA文库的效果,确定了以生物素-链霉亲和素磁珠分离方法制备ssDNA为优选方法。ssDNA文库扩增为双链DNA(dsDNA)文库的PCR反应条件为:退火温度50℃,循环数20次。不对称PCR退火温度为50℃,最佳循环数为30次,引物Ⅰ与引物Ⅱ的比例为80∶1。此反应条件下,ssDNA文库PCR扩增的条带清晰、稳定、特异性高。生物素-链霉亲和素磁珠法为有效筛选特异性强的玉米赤霉烯酮单克隆抗体适配子的优选技术方案,为适配子SELEX筛选奠定基础。