The bacteria strains in this study.

Melioidosis, caused by Burkholderia pseudomallei, is a tropical disease known for its long incubation period and high mortality rate. However, the traits of this “great imitator” present significant challenges for clinical diagnosis and pose a serious threat to populations in epidemic regions. A rapid, accurate, and low environment requirement diagnostic method is needed to enable timely diagnoses. Here, we developed a strategy for point-of-care testing (POCT) using loop-mediated isothermal amplification (LAMP) with RNase HII for efficient detection of B. pseudomallei, through real-time fluorescence analysis or lateral flow strip assay (LFSA). Briefly, RNase HII-based cleavage dual-functional probe LAMP (RCDP-LAMP) utilizes RNase HII cleave the “rG” from the probe which hybridized with target. FAM group was cleaved, detaching from BHQ1, and exciting fluorescence or Biotin group on the same side as BHQ1 was used as the target captured in LFSA for the results. The limit of detection (LOD) for is 50 fg/reaction. In summary, RCDP-LAMP presents a versatile tool for detecting B. pseudomallei in both professional clinical laboratories and POCT scenes, suitable for large-scale screening efforts. This approach is essential for controlling costs in primary healthcare units and significantly enhances the prevention and management of the severely neglected tropical disease.