Development of a point-of-care testing (POCT)-use paper-based device for recombinase polymerase amplification (RPA)-based bioassays-: Demonstration of the detection of Neisseria gonorrhoeae

The point of care test (POCT) of pathogens is critical for various applications. Nevertheless, the current immunological- or polymerase chain reaction (PCR)-based assays might not meet the requirements for POCT (e.g., ASSURED: affordable, sensitive, specific, user-friendly, rapid, equipment-free, and delivered proposed by the World Health Organization). To address this issue, this study proposed a paper-based device within which the functions of nucleic acid isolation, recombinase polymerase amplification (RPA)-based nucleic acid amplification, and lateral flow immunoassays were integrated. For RPA, a disposable hand warmer capable of providing 45 °C temperature was attached underneath the paper-based device for achieving equipment-free bioassays. Unlike the other paper-based devices, the presented device maintains the working area for reagent loading at a fixed area to largely simplify the operation procedures making a bioassay user-friendly. In this study, its working performances were evaluated. Results demonstrated that the detection sensitivity was 10 copies of genomic DNA under 10 min RPA reaction time, which was 10 times lower than that (100 copies) based on the conventional RPA-based assays. Also, its application for the detection of Neisseria gonorrhoeae in urine was successfully demonstrated. Results revealed it was able to detect N. gonorrhoeae with detection sensitivity of 1 CFU/ml. Overall, this study presented a paper-based device capable of performing isothermal nucleic acid amplification tests in a user-friendly, low-cost, equipment-free, rapid (30- 35 min) and high detection sensitivity (10 copies of DNA, or 1 CFU/ml of bacteria) manner. These features make it suitable for the applications in POCT, home care, or resource-limited regions.