Paper-based speedy separation of amplified DNA (PASS-DNA): Potential for molecular point-of-care testing

Abstract

Point-of-care testing (POCT) is a simple, rapid and cost-efficient diagnostic method in settings where clinical resources are limited, developing countries for instance, and an essential tool for controlling infectious diseases. Previously, we developed a centrifugal-based separation-free visual detection system (SPIN-DNA) for human papillomavirus (HPV) DNA. However, this gel column-based detection system requires a centrifugation device to separate DNA-bound beads from free beads. It would be more feasible in resource-limited settings to employ a smaller force, such as a capillary force, for lateral flow assay. For this purpose, here we describe a paper-based method for separating amplified DNA that we developed. Visualization of amplified DNA to the naked eye using magnetic beads was achieved, without additional centrifugation, addition of capturing DNA probes, incubation time, or dilution. This simple visualized detection method of target DNA (PASS-DNA) was applied for HPV DNA in this study. The detection sensitivity of PASS-DNA was at least 101 copies/mL of HPV DNA, and the results of PASS-DNA showed perfect agreement with those obtained using the Roche Cobas 4800 HPV test (n = 30). No prolonged incubation times or washing steps are required with PASS-DNA; rather, the method involves one mixing step followed by loading the solution onto the paper. In addition, no absorbent pad is needed to provide a capillary driving force. This paper-based DNA detection system could accelerate the development of molecular POCT with further improvements and integration with sample preparation and DNA isolation and amplification in resource-limited settings.