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Published on: February 26, 2018
Direct detection from clinical sputum samples to differentiate live and dead Mycobacterium Tuberculosis
Jie Lu1, Huiwen Zheng1, Ping Chu1
1Beijing Key Laboratory for Pediatric Diseases of Otolaryngology, Head and Neck Surgery, MOE Key Laboratory of Major Diseases in Children, Beijing Pediatric Research Institute, Beijing Children's Hospital, National Center for Children's Health, Capital Medical University, Beijing, China.
Background:
In this study, we aimed to optimize the condition of propidium monoazide (PMA) treatment for direct detection of Mycobacterium tuberculosis (MTB) from clinical specimens.
Methods:
The light exposure time, dark incubation time, bacterial load, and PMA concentration were varied to determine the optimal condition of PMA treatment.
Results:
Overall, the maximum ΔCq value was observed in the group receiving a light exposure time of 20 minutes, which was significantly higher than the others (P < 0.05). The prolongation of dark incubation time seemed more likely to result in greater ΔCq value, and the ΔCq values were 2.0, 4.1, 6.5, 10.1, and 12.7 cycles under dark incubation time of 10, 20, 40, 60, and 120 minutes, respectively. Alternatively, the 4+ samples exhibited favorable detection results at the application of 104 -fold dilution by PMA assay with Cq values higher than 35 cycles. Further evaluation revealed that the PMA assay showed an accordance rate of 98.0% (98/100) among clinical sputa.
Conclusions:
we develop an acceptable method to directly identify the live bacteria from sputum samples. Our data demonstrate that the dark incubation plays a crucial role in the efficacy of PMA treatment for MTB.
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