Tuberculosis (TB) remains one of the major infectious diseases worldwide. The pathogenic bacterium, Mycobacterium tuberculosis (M.tb), continuously evolves strains carrying drug-resistance genes, thus posing a growing challenge to TB prevention and treatment. We report a diagnostic system that uses a molecular beacon probe and an assistant strand as the core to simultaneously interact with an M.tb-specific fragment (in IS6110) and a single nucleotide substitution (SNS)-encoded segment (in rpoB) associated with drug resistance. A single fluorescent output in three-tiered levels was produced for combinatorial interpretations based on formation of a four-way DNA junction (4WJ). The SNS caused the 4WJ to partially dissociate, thus resulting in medium-level fluorescence. By contrast, high- and low-level fluorescence, represented the complete complementary complex and absence of either targeted fragments, respectively. Manipulating the length of the analyte-binding arm realized the medium output. The thermodynamics and kinetics of 4WJ construction were investigated to maximize the tiered-output performance. Biocatalytic amplification driven by the Klenow Fragment and Nt.AlwI was incorporated into the method to enhance the signal 64-fold and ensure long-term stability of the three-tiered output. The detection accuracy of the sensing system was verified using unpurified amplicons with templates of extracted DNA and boiled bacterial solutions. The tiered-output mechanism was usable at bacterial loads ranging from 4 × 100 to 4 × 103 CFU per reaction. The interference caused by nontuberculous mycobacteria was minimal. The results demonstrated the integrity of the sensing method as an alternative strategy for rapid screening of M.tb and detecting rifampin-resistance.

中文翻译：

---

结核病诊断的三级输出同时检测遗传片段和单核苷酸突变

结核病 (TB) 仍然是世界范围内的主要传染病之一。病原菌结核分枝杆菌（M.tb）不断进化出携带耐药基因的菌株，从而对结核病的预防和治疗提出了越来越大的挑战。我们报告了一种诊断系统，该系统使用分子信标探针和辅助链作为核心，同时与 M.tb 特异性片段（在 IS6110 中）和单核苷酸取代（SNS）编码的片段（在 rpoB 中）相互作用。耐药性。基于四向 DNA 连接 (4WJ) 的形成，产生了三层水平的单一荧光输出，用于组合解释。SNS 导致 4WJ 部分解离，从而产生中等水平的荧光。相比之下，高强度和低强度的荧光，分别代表完整的互补复合物和不存在任一目标片段。操纵分析物结合臂的长度实现了中等输出。研究了 4WJ 结构的热力学和动力学，以最大限度地提高分层输出性能。由 Klenow Fragment 和 Nt.AlwI 驱动的生物催化放大被纳入该方法，以将信号增强 64 倍并确保三层输出的长期稳定性。使用未纯化的扩增子与提取的 DNA 和煮沸的细菌溶液模板来验证传感系统的检测准确性。分层输出机制可用于每个反应 4 × 100 到 4 × 103 CFU 的细菌负荷。非结核分枝杆菌引起的干扰很小。