Rapid Isothermal DNA Amplification in Microchambers Detected by Fluorescence RNA Aptamer Transcription

Objectives: Rapid detection and quantification of nucleic acids are essential for on-site diagnosis of pathogens. To provide an alternative to current methods that require bulky instruments and long reaction times, we developed a digital nucleic acid amplification method suitable for point-of-care applications. Methods: The method combines compartmentalization in micrometer-sized microchambers with recombinase polymerase amplification (RPA) and the Mango fluorescent aptamer system. Fluorescence microscopy was used to acquire images of microchambers. Single molecules of target DNA sequences were detected as fluorescence-positive chambers in the image and quantified by counting these chambers. Results: Detection and quantification were achieved within 8 and 22 min, respectively. The measurable concentration range was approximately 4 fM to 40 pM, demonstrating a wide dynamic range. Furthermore, the successful detection of five different pathogen-derived DNA sequences confirmed the versatility of the approach. Conclusions: Because the reaction proceeds isothermally within a compact microdevice, the system requires minimal instrumentation. These features make it a promising platform for nucleic acid measurement in point-of-care testing.