Development of a chemical precipitated-LAMP assay for the diagnosis of knowlesi malaria

A visualized air-dried precipitated loop-mediated isothermal amplification (ADP-LAMP) master mix, incorporating hydroxynapthol blue dye, was developed for the detection of Plasmodium knowlesi. The ADP-LAMP assay was designed to overcome the reliance of LAMP assays on cold-chain transportation for liquid reagents. This study analyzed 80 P. knowlesi and 40 non-P. knowlesi human blood samples collected from the district hospitals. Samples were confirmed by microscopy and nested PCR prior to ADP-LAMP method. The amplification buffer, enzyme and primers involved in ADP-LAMP were chemically precipitated followed by spin-drying into room-temperature stabled pellets. The ADP-LAMP assay generated result which could be visualized by naked eye without the use of specialized equipment and the limit of detection was found to be 10 copies/μL of P. knowlesi. The assay demonstrated high specificity, with no cross-reactivity against other human malaria species or human genomic DNA. When evaluated on clinical samples, the ADP-LAMP assay demonstrated 100% specificity and sensitivity with standard nested PCR and delivers results within 45 minutes at 65°C. This ADP-LAMP assay could be served as an alternative diagnostic tool in malaria endemic areas.