Zhang Yue, Liang Xiuqiong, Yao Juan, Liu Wei, Zhang Nan, Zhang Zhang, Xia Qianfeng
NHC Key Laboratory of Tropical Disease Control, School of Tropical Medicine, Hainan Medical University, Haikou, Hainan, China.
Nanobiosensing and Microfluidic Point-of-Care Testing, Key Laboratory of Luzhou, Department of Clinical Laboratory, The Affiliated Traditional Chinese Medicine Hospital, Southwest Medical University, Luzhou, Sichuan, China.
PLoS Negl Trop Dis. 2025 Jun 4;19(6):e0013109. doi: 10.1371/journal.pntd.0013109. eCollection 2025 Jun.
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.
类鼻疽病由嗜麦芽窄食单胞菌引起，是一种热带疾病，以其潜伏期长和死亡率高而闻名。然而，这种“伪装大师”的特性给临床诊断带来了重大挑战，并对流行地区的人群构成严重威胁。需要一种快速、准确且对环境要求低的诊断方法来实现及时诊断。在此，我们开发了一种即时检测（POCT）策略，使用基于核糖核酸酶HII的环介导等温扩增（LAMP）技术，通过实时荧光分析或侧向流动试纸条检测（LFSA）来高效检测嗜麦芽窄食单胞菌。简而言之，基于核糖核酸酶HII的切割双功能探针LAMP（RCDP-LAMP）利用核糖核酸酶HII从与靶标杂交的探针上切割掉“rG”。荧光素家族（FAM）基团被切割，从黑洞猝灭剂1（BHQ1）上脱离，从而激发荧光，或者将与BHQ1在同一侧的生物素基团用作LFSA中捕获靶标的物质以得出结果。检测限为50 fg/反应。总之，RCDP-LAMP为在专业临床实验室和POCT场景中检测嗜麦芽窄食单胞菌提供了一种通用工具，适用于大规模筛查工作。这种方法对于控制基层医疗单位的成本至关重要，并显著加强了对这种严重被忽视的热带疾病的预防和管理。
