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 × 102 to 5.0 × 105 CFU mL-1, and the detection limit is 3.8 × 102 CFU mL-1 (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.
Keywords: Adenosine triphosphate; Bioluminescence; Magnetic particles; Mycobacteriophage; Mycobacterium smegmatis.
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