Establishment of a visual mixed-dye loop mediated isothermal amplification assay for detecting Plasmodium knowlesi

Colorimetric detection of loop-mediated isothermal amplification (LAMP) amplicons is one of the easiest and most direct methods among all detection techniques. However, not all indicators exhibit a noticeable colour change for both positive and negative reactions. In this study, we present a novel and visualized dual colour LAMP assay for the detection of Plasmodium knowlesi. The assay utilizes two types of dyes (phenol red and hydroxynaphthol blue) to facilitate a clear and distinguishable colour change upon successful amplification. The colour changes generated by the current single dye LAMP is low contrast and sometimes not significant. Thus, we propose a new approach to enlarge the variation colour range as well as improve its suitability for naked-eye observation. The dual colour system enhances the visual readout of the assay, thereby reducing false positives and negatives. To evaluate the performance of the mixed-dye LAMP, we test it with 55 clinical samples. By using nested PCR as the reference method, dual-colour LAMP showed 100 % sensitivity (95 % confidence interval (CI): 91.9-100 %) and 100 % specificity (95 % CI: 78.2-100 %). This mixed-dye LAMP assay offers a rapid, cost-effective, and user-friendly diagnostic tool for P. knowlesi malaria, particularly suitable for resource-limited settings.