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

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.

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。

艰难梭菌是引起院内感染的主要病原体之一，快速、灵敏地诊断艰难梭菌感染具有重要意义。目前，临床上常用的诊断方法存在敏感性低、耗时久等缺陷。本研究利用ＳＥＬＥＸ技术筛选了艰难梭菌毒素Ｂ特异性适体，构建了基于磁分离和适体介导的比色法。利用捕获探针将ＨＲＰ标记的适体固定在磁珠上。当有靶物质存在时，适体特异性地识别并结合TcdB，破坏ＤＮＡ杂交，ＨＲＰ标记的适体从磁珠上脱离，减少ＨＲＰ的催化活性，导致吸光强度降低。由于吸光度与靶物质有线性关系，因此通过检测吸光度的变化可以定量检测艰难梭菌毒素Ｂ的浓度，检测限为5ng。本研究可为临床上艰难梭菌感染的早期诊断提供有力的技术支持，对控制医院感染、促进人民身体健康具有重要意义。