Department of Convergence Medicine, Asan Medical Institute of Convergence Science and Technology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea.
Academy of Medical Engineering and Translational Medicine, Tianjin University, Tianjin Key Laboratory of Brain Science and Neural Engineering, Tianjin, China.
Sci Rep. 2019 Nov 8;9(1):16374. doi: 10.1038/s41598-019-52922-y.
Here, we describe a simple, universal protocol for use in nucleic acid testing-based pathogen diagnostics, which requires only hand-powered sample preparation, including the processes of pathogen enrichment and nucleic acid isolation. The protocol uses low-cost amine-functionalized diatomaceous earth with a 1-μm Teflon filter as a reaction matrix in both stages of the process, using homobifunctional imidoesters. Using a simple syringe as a pump, the capture efficiency for a large sample volume (<50 mL) was enhanced by up to 98.3%, and the detection limit was 1 CFU/mL, 100-fold better than that of common commercial nucleic acid isolation kit. This protocol can also be combined with commercialized 96-well filter plates for robust sample preparation. Our proposed system is robust, simple, low-cost, universal, and rapid (taking <20 min), and it works regardless of the ambient environment and sample pretreatment, requiring no electricity or instruments. Its benefits include the simplicity of producing its components and its ease of operation, and it can be readily integrated with other assays for point-of-care diagnostics.
在这里，我们描述了一种简单、通用的基于核酸检测的病原体诊断协议，该协议仅需手动进行样品制备，包括病原体富集和核酸分离过程。该协议使用低成本的胺功能化硅藻土和 1μm 的聚四氟乙烯过滤器作为两个过程中的反应基质，使用同双功能亚氨酸酯。使用简单的注射器作为泵，通过该方法可以将大容量样品（<50mL）的捕获效率提高多达 98.3%，检测限为 1 CFU/mL，比常见的商用核酸分离试剂盒灵敏 100 倍。该方案还可以与商业化的 96 孔滤板结合使用，用于可靠的样品制备。我们提出的系统具有强大、简单、低成本、通用和快速（<20 分钟）的特点，无论环境条件和样品预处理如何，它都可以正常工作，无需电力或仪器。其优点包括其组件的简单生产和易于操作，并且可以与其他即时诊断检测方案集成。
