Early diagnosis of tuberculosis can dramatically reduce both its transmission and the associated death rate. The extremely slow growth rate of the causative pathogen, Mycobacterium tuberculosis (Mtb), however, makes this challenging at the point of care, particularly in resource-limited settings. Here we report the use of BlaC (an enzyme naturally expressed/secreted by tubercle bacilli) as a marker and the design of BlaC-specific fluorogenic substrates as probes for Mtb detection. These probes showed an enhancement by 100–200 times in fluorescence emission on BlaC activation and a greater than 1,000-fold selectivity for BlaC over TEM-1 β-lactamase, an important factor in reducing false-positive diagnoses. Insight into the BlaC specificity was revealed by successful co-crystallization of the probe/enzyme mutant complex. A refined green fluorescent probe (CDG-OMe) enabled the successful detection of live pathogen in less than ten minutes, even in unprocessed human sputum. This system offers the opportunity for the rapid, accurate detection of very low numbers of Mtb for the clinical diagnosis of tuberculosis in sputum and other specimens.
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This work was supported by grant 48523 from the Bill and Melinda Gates Foundation, the Welch Foundation grant no. A-0015 and NIH P01 68135 for TB Structural Genomics. We thank Bob Fader in the Scott & White Memorial Hospital (Temple, Texas) for providing sputum samples from cystic fibrosis patients.
The authors declare no competing financial interests.
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Xie, H., Mire, J., Kong, Y. et al. Rapid point-of-care detection of the tuberculosis pathogen using a BlaC-specific fluorogenic probe. Nature Chem 4, 802–809 (2012). https://doi.org/10.1038/nchem.1435
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