Label-free citrate-stabilized silver nanoparticles-based highly sensitive, cost-effective and rapid visual method for differential detection of Mycobacterium tuberculosis and Mycobacterium bovis

Tuberculosis poses a global health challenge, demanding improved diagnostics and therapies. Distinguishing between Mycobacterium tuberculosis (M. tb) and Mycobacterium bovis (M. bovis) infections holds critical "One Health" significance due to zoonotic nature of these infections and inherent resistance of M. bovis to pyrazinamide, a key part of Directly Observed Treatment, Short-course (DOTS) regimen. Furthermore, most of the currently used molecular detection methods fail to distinguish between the two species. To address this, our study presents an innovative molecular-biosensing strategy. We developed a label-free citrate-stabilized silver nanoparticle aggregation assay, offering sensitive, cost-effective, and swift detection. For molecular detection, genomic markers unique to M. tb and M. bovis were targeted using species-specific primers. In addition to amplifying species-specific regions, these primers also aid detection of characteristic deletions in each of the mycobacterial species. Post polymerase chain reaction (PCR), we compared two highly sensitive visual detection methods with respect to the traditional agarose gel electrophoresis. The paramagnetic bead-based bridging flocculation assay, successfully discriminates M. tb from M. bovis with a sensitivity of ~40 bacilli. The second strategy exploits citrate-stabilized silver nanoparticle which aggregates in the absence of amplified dsDNA on addition of sodium chloride (NaCl). This technique enables precise, sensitive and differential detection of as few as ~4 bacilli. Our study hence advances tuberculosis detection, overcoming challenges of M. tb and M. bovis differentiation offering a quicker alternative to time-consuming methods.