Recombinase-Aided Loop-Mediated Isothermal Amplification on Human Plasmodium knowlesi

Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification technique that can amplify specific nucleic acids at a constant temperature (63-65°C) within a short period (<1 hour). In this study, we report the utilization of recombinase-aided LAMP to specifically amplify the 18S sRNA of Plasmodium knowlesi. The method was built on a conventional LAMP assay by inclusion of an extra enzyme, namely recombinase, into the master mixture. With the addition of recombinase into the LAMP assay, the assay speed was executed within a time frame of less than 28 minutes at 65°C. We screened 55 P. knowlesi samples and 47 non-P. knowlesi samples. No cross-reactivity was observed for non-P. knowlesi samples, and the detection limit for recombinase-aided LAMP was one copy for P. knowlesi after LAMP amplification. It has been reported elsewhere that LAMP can be detected through fluorescent readout systems. Although such systems result in considerable limits of detection, the need for sophisticated equipment limits their use. Hence, we used here a colorimetric detection platform for the evaluation of the LAMP assay's performance. This malachite green-based recombinase-aided LAMP assay enabled visualization of results with the naked eye. Negative samples were observed by a change in color from green to colorless, whereas positive samples remained green. Our results demonstrate that the LAMP assay developed here is a convenient, sensitive, and useful diagnostic tool for the rapid detection of knowlesi malaria parasites. This method is suitable for implementation in remote healthcare settings, where centralized laboratory facilities, funds, and clinicians are in short supply.