Rapid detection of zoonotic malaria using room-temperature stable and ready-to-use colorimetric LAMP reagents

Traditional loop-mediated isothermal amplification (LAMP) assays rely on a cold chain to preserve the stability of LAMP reagents during storage and transport. This requirement restricts the application of LAMP-based methods in resource-limited settings. To overcome this challenge, we developed a room-temperature stable, ready-to-use dried format of the LAMP diagnostic tool for detecting zoonotic malaria. In this study, we report the use of LAMP for detecting five zoonotic malaria species including Plasmodium fieldi, as there have been no previous publications on LAMP for fieldi to date. The reaction mixture was spin-dried in a vacuum container and stored with silica gel at room temperature before used. The detection limits were 1 copy/µL for P. cynomolgi, P. coatneyi, P. fieldi, and P. inui, while the detection limit for P. knowlesi was 10 copies/µL. Results can be visually observed through color changes, without the need for specialized equipment. Positive samples were indicated in yellow while negative reactions remained pink. The dehydrated LAMP reagents can be kept at room temperature for 15 days. Combination of the advantages of the raffinose, the dehydrated LAMP assay develop here is an ideal diagnostic tool in resource-limited settings where malaria is endemic.