DNA extraction from bacteria using a gravity-driven microcapillary siphon

Abstract

Nucleic acid amplification tests (NAATs) are used in many applications ranging from human diagnostics to environmental monitoring. However, currently NAATs rely on access to bulky equipment and laboratory facilities. In particular, DNA extraction remains a key limiting step, with current methods depending on large devices and extensive manual pipetting. The design of portable NAAT devices and wider access to molecular testing is therefore of critical scientific and medical importance. Here we report the first application of a gravity-driven microcapillary siphon for the extraction of bacterial DNA. Making use of magnetic DNA-binding beads and an external magnet, capturing, washing and DNA elution were performed in 10-bored, 200 μm internal diameter siphons using passive flow, without the need for external pumps nor other specialised equipment. The method was successfully evaluated using real-time qPCR, from where we observed a linear relationship between colony forming units (CFUs) and threshold cycle (Ct) for E. coli spiked in several matrices relevant for pathogen analysis: PBS buffer, sheep blood, and river water. Importantly, this method was not only cost-effective but also outperformed the standard manual protocol, returning higher recovery efficiency (>90% vs. 52%) and better removal of assay inhibitors, which we believe linked to the higher shear rates and short diffusion distances obtained in the flow-through, microcapillary siphons. In addition, we have shown microcapillary siphons are reusable without detectable cross-contamination between consecutive DNA extractions. These findings demonstrate that high quality DNA can be isolated without access to expensive equipment or complex fluid handling. Future work will explore integration with isothermal amplification methods for development of a fully integrated, passive-flow devices for portable NAATs.