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.
Bibliographical noteFunding Information:
This study was supported by a grant from the Korea Health Technology R & D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (HI16C-0272-010016), and supported by the Ministry of Science, ICT, and Future Planning (MSIP) through the National Research Foundation of Korea (NRF) (2017R1A2B4005288), and also supported by grants from Asan Institute for Life Sciences (2019-7034), Republic of Korea.
© 2019, The Author(s).
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