基于毛细管阵列的超级细菌微全基因分析系统的构建

Superbug is a new subgroup bacteria with the New Delhi metallo-β-lactamase 1 (NDM-1), a broad-spectrum-lactamase that is able to inactivate nearly all-lactams. This type of pathogens is easy to spread, and usually with high fatality rate. Therefore, the development of a rapid and effective detection method is of great clinically important significance to the prevention and treatment of superbug. Based on our previous work, this project plans to develop a capillary-array based micro total analysis system for rapid genetic detection of superbugs. This microsystem proposed features: 1. Integration of the entire process that related to loop-mediated isothermal amplification(LAMP)-based genetic testing by using a capillary droplet method; 2. Parallel analysis of samples using capillary array; 3. Combination of transparent indium-tin oxide glass as heater and a blue-light LED array as detection, to reduce equipment size and simplify the analysis operation. This project includes the development of the micro total analysis system, the analytical reagents and the analytical methods. Our study is to systematically test performances of the micro total analysis system, and to evaluate feasibility of the microsystem by detecting batch of clinical samples. Through optimizations of system structure and analytical approaches, this work aims to achieve rapid detection of this kind of bacteria. The micro total analysis has advantages of simplicity, rapidness, moderate throughput and low cost, thus is expected to address the urgent need for rapid detection of superbug in clinic work. Furthermore, the micro total analysis system can be developed into an universal platform for rapid genetic detection of various kinds of pathogens.

超级细菌感染病死率高且易于传播，临床防治形势严峻，发展快速有效的检测方法对临床上超级细菌的防治具有重要意义。在前期工作基础上，本研究计划发展一种基于毛细管阵列的微全基因分析系统，用于临床超级细菌快速检测。该系统具有以下特点：1.在毛细管中借助液滴操作，集成环介导等温扩增（LAMP）基因检测的整个过程；2.利用毛细管阵列实现平行样品分析；3.使用透明的氧化铟锡(ITO)玻璃作为加热器，以蓝光LED阵列作为检测光源，二者的结合有利于减小仪器尺寸和简化分析操作。利用自行开发的分析试剂，发展一整套用于超级细菌检测的检测方法。研究将对这种微全分析方法进行系统评价，并以批量临床样品进行系统的可行性测试。通过必要的系统结构以及分析方法优化，最终实现超级细菌的快速检测。这种微全分析方法简单快速、通量适中且成本低廉，有望解决临床工作中超级细菌快速检测的迫切需要并有望发展成为通用型病原微生物快速检测平台。

超级细菌NDM-1感染病死率高且易于传播，临床防治形势严峻，发展快速有效的检测方法对临床上超级细菌的防治具有重要意义。本研究发展了一种基于毛细管阵列的微全基因分析系统，用于临床超级细菌NDM-1快速检测。完成了以下工作：1.按照功能要求设计系统外部结构如温控装置、电磁控制装置及信号检测装置；系统内部结构完善等如毛细管中的液滴形成与运输、磁珠的固定与振荡、DNA提取、LAMP扩增及实时监测扩增信号检测等功能。2.针对NDM-1细菌作了以下优化：前处理步骤(菌体捕获、DNA提取)、LAMP引物优化，毛细管中LAMP体系与反应条件优化、LAMP扩增信号检测、标准品曲线制作等。考察样本检测灵敏度和特异性、考察临床实际分离细菌标本的检测能力，初步评价其临床应用价值。这种微全分析方法简单快速、通量适中且成本低廉，有望解决临床工作中NDM-1细菌快速检测的迫切需要并有望发展成为通用型病原微生物快速检测平台。