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.

由假马利伯克霍尔德菌（Burkholderia pseudomallei）引发的类鼻疽（Melioidosis）是一种热带传染病，以漫长的潜伏期与极高的病死率为典型特征。该病原体素有“伟大的模仿者”之称，其生物学特性给临床诊断带来了极大挑战，也对流行区域的人群健康构成严重威胁。目前亟需一种快速、精准且环境适配要求低的诊断方法，以实现及时确诊。本研究开发了一种基于核糖核酸酶HII（RNase HII）的环介导等温扩增（LAMP）即时检测（POCT）策略，可通过实时荧光分析或侧向流试纸条检测（LFSA）实现假马利伯克霍尔德菌的高效检测。具体而言，基于核糖核酸酶HII切割的双功能探针环介导等温扩增（RCDP-LAMP）技术，利用RNase HII切割与靶序列杂交的探针上的“rG”位点：羧基荧光素（FAM）基团被切割后与黑洞猝灭剂1（BHQ1）分离，从而激发荧光信号；而与BHQ1同侧的生物素（Biotin）基团则可作为靶标捕获位点，用于侧向流试纸条检测的结果判读。该方法的检测限（LOD）可达50 fg/反应体系。综上，RCDP-LAMP技术是一款通用的检测工具，既可应用于专业临床实验室，也适用于即时检测场景，可用于大规模人群筛查。该方案有助于基层医疗卫生机构控制检测成本，可显著提升这一被严重忽视的热带传染病的防控与管理水平。