Hydroxynaphthol Blue-Based Recombinase Polymerase Amplification for the Detection of Plasmodium knowlesi

Background: A rapid and easy-to-use diagnostic tool is essential for the accurate diagnosis of malaria. Recombinase polymerase amplification (RPA) is well suited for on-site detection due to its rapid turnaround, high sensitivity, and operation at low temperatures. However, result visualization commonly relies on fluorescence probes, which require specialized equipment, or lateral flow strips, which add to the overall assay cost. Methods: In this study, we developed a hydroxynaphthol blue (HNB)-based RPA assay for the visual detection of Plasmodium knowlesi . As well, we attempted to performed RPA without DNA isolation via a commercial kit. This method utilizes a simple DNA preparation method involving dilution and heat treatment of whole blood, eliminating the need for commercial DNA extraction kits. The entire extraction process was completed in less than 10 mins. Within 20 mins of incubation at 37°C, amplification was confirmed by a visible color change, enabling simple, equipment-free interpretation. Results: The assay demonstrated high analytical sensitivity, with a limit of detection of 0.005 parasites/µL. No cross-reactivity was observed with other Plasmodium species, indicating high specificity of the HNB-RPA assay. Conclusions: This assay provides a rapid, sensitive, and cost-effective diagnostic approach, suitable for resource-limited and field settings.