基于核酸适体和便携式血糖仪的艰难梭菌毒素A快速检测新方法的研究

项目名称： 基于核酸适体和便携式血糖仪的艰难梭菌毒素A快速检测新方法的研究

项目编号： No.81501834

项目类型： 青年科学基金项目

立项/批准年度： 2016

项目学科： 医药、卫生

项目作者： 罗鹏

作者单位： 重庆医科大学

项目金额： 18万元

中文摘要： 艰难梭菌(Clostridium difficile)是引起院内感染性腹泻的主要病原菌，可导致严重并发症。目前艰难梭菌检测方法特异性差、操作繁琐，不能满足临床快速诊断和院感监控的需要。适体(Aptamer)是能特异性结合靶分子的核酸序列，在生物分析过程中起重要作用。在前期研究中我们筛选出一条能识别艰难梭菌毒素A(Toxin A,TOA)的适体，并基于该适体构建了可快速检测TOA的生物传感器。但是，该方法需要专业的电化学工作站，影响了它的临床实际应用。为了弥补该方法的不足，本研究拟将所筛选的TOA适体偶联海藻糖酶，与嗜热脂肪芽孢杆菌和枯草芽孢杆菌合成的纳米金包裹的四氧化三铁构成生物传感界面，再利用海藻糖酶能特异催化海藻糖生成葡萄糖的属性，应用便携式血糖仪定量TOA，创建一种方便、快速检测艰难梭菌的新方法，对控制医院感染具有重要意义。

中文关键词： 艰难梭菌；适体；血糖仪；生物合成；金纳米粒子

英文摘要： Clostridium difficile is one of the most predominant pathogens of nosocomial intestinal infections, which can cause antibiotic-associated diarrhea or pseudo-membranous colitis in humans and animals. Currently, conventional methods such as microbiological culture and ELISA are being used for determination of Clostridium difficile. However, these methods are usually insensitive and time-consuming,which can not contribute to the rapid diagnosis and the control of nosocomial infections. An aptamer is a synthetic single-stranded DNA or RNA oligonucleotide that can specifically bind to target molecules,which has played an important role in biochemical analyses. In our previous research, An aptamer against toxin A of Clostridium difficile was selected using systematic evolution of ligands by exponential enrichment (SELEX) method, and then a sensitive aptasensor was developed to detect TOA. However, the above aptasensor still required laboratory-based devices that were not really available to the clinical work. To meet the challenge, we propose the use of trehalase, which is an enzyme catalyzing the hydrolysis of trehalose into glucose, to link glucose detection to the detection of TOA, and use the portable glucose meter to quantify TOA. The sensing interface was composed of aptamer-conjugated trehalase and Fe3O4 nanospheres encapsulated with gold nanoparticles synthesized by Bacillus stearothermophilus and Bacillus subtilis. This study can provide a rapid and sensitive diagnostic procedure for Clostridium difficile and is important for controlling the hospital infection caused by Clostridium difficile.

英文关键词： clostridium difficile;aptamer;glucose meter;biological synthesis;gold nanoparticles