Abstract
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.
Highlights
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
amine-functionalized diatomaceous earth (ADE)’s nano-porous structure gives it strong absorption capability and it has an ultra-high reaction area, which contributes to its enhanced performance[34]
A syringe filter was utilized to trap ADE and its attached pathogens, so the enrichment process could be performed by mixing the dimethyl suberimidate (DMS) and www.nature.com/scientificreports
Summary
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. They generally involve intricate pretreatments (such as pre-fabrication or sample centrifugation), which require laboratory-based procedures involving multiple steps, skilled technicians, and specific instruments[12] These drawbacks limit the application of NAT to point-of-care testing (POCT), especially in resource-limited settings. When using conventional FTA card-based assays (including commercial kits, e.g., Whatman FTA cards), there are difficulties when faced with low-concentration analytes (femtomolar or nanomolar) and small volumes (1–1000 μL) within complex biological media (e.g., whole blood samples)[27]. They are not offered to the large-volume sample treatment by the intrinsic limitations, such as spotting-based sample loading process and small sample loading disk
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