Yue Xin, Huang Yu, Zhang Yu, Ouyang Hui, Xie Jianping, Fu Zhifeng
Key Laboratory of Luminescence Analysis and Molecular Sensing (Ministry of Education), College of Pharmaceutical Sciences, Southwest University, Chongqing, 400716, China.
State Key Laboratory Breeding Base of Eco-Environment and Bio-Resource of the Three Gorges Area, Key Laboratory of Eco-environments in Three Gorges Reservoir Region, Ministry of Education, School of Life Sciences, Southwest University, Chongqing, 400715, China.
Anal Chim Acta. 2021 Feb 8;1145:17-25. doi: 10.1016/j.aca.2020.12.009. Epub 2020 Dec 8.
Mycobacterium tuberculosis (M. tuberculosis), the causative agent of tuberculosis, ranks one of the most dangerous pathogens for its large deaths toll. Due to its characteristic extremely slow growth, the conventional culture-based protocol cannot meet the requirement for the efficient diagnosis of M. tuberculosis-induced tuberculosis. With our previously isolated mycobacteriophage SWU1, we tried to develop a mycobacteriophage-based protocol for detecting Mycobacterium genus. In this work, Mycobacterium smegmatis (M. smegmatis) was used as a model due to its similar physiological features as pathogenic M. tuberculosis, much faster growth and nonpathogenic property. Mycobacteriophage SWU1-functionalized magnetic particles (SWU1-MPs) were used as highly efficient separation carriers for the viable host M. smegmatis. After a replication cycle of approximate 60 min, the cells of M. smegmatis were disrupted by the progeny mycobacteriophages to release intracellular adenosine triphosphate (ATP). The bioluminescent (BL) signal of released ATP was collected to quantitate the amount of M. smegmatis. For the developed protocol, the detection range is 5.0 × 10 to 5.0 × 10 CFU mL, and the detection limit is 3.8 × 10 CFU mL (S/N = 3). Furthermore, the protocol can exclude the potential interference of 3 non-pathogenic mycobacteria and 6 other bacterial species. It has been successfully applied to quantitate M. smegmatis in human urine, human saliva, and human serum. The results demonstrate its application potential for a simple, fast, and specific diagnosis of M. tuberculosis infection.
结核分枝杆菌是结核病的病原体，因其造成大量死亡而成为最危险的病原体之一。由于其生长极其缓慢的特性，传统的基于培养的检测方法无法满足高效诊断结核分枝杆菌所致结核病的需求。利用我们之前分离的分枝杆菌噬菌体SWU1，我们试图开发一种基于分枝杆菌噬菌体的检测分枝杆菌属的方法。在这项工作中，耻垢分枝杆菌因其生理特征与致病性结核分枝杆菌相似、生长速度快得多且无致病性，被用作模型。分枝杆菌噬菌体SWU1功能化的磁性颗粒（SWU1-MPs）被用作活宿主耻垢分枝杆菌的高效分离载体。经过约60分钟的复制周期后，耻垢分枝杆菌细胞被子代分枝杆菌噬菌体破坏，释放出细胞内三磷酸腺苷（ATP）。收集释放的ATP的生物发光（BL）信号以定量耻垢分枝杆菌的数量。对于所开发的方法，检测范围为5.0×10至5.0×10 CFU/mL，检测限为3.8×10 CFU/mL（S/N = 3）。此外，该方法可以排除3种非致病性分枝杆菌和6种其他细菌的潜在干扰。它已成功应用于定量人尿液、人唾液和人血清中的耻垢分枝杆菌。结果证明了其在结核分枝杆菌感染的简单、快速和特异性诊断中的应用潜力。
