Label-Free Citrate-Stabilized Silver Nanoparticles-Based, Highly Sensitive, Cost-Effective, and Rapid Visual Method...

Tuberculosis poses a global health challenge, and it demands improved diagnostics and therapies. Distinguishing between Mycobacterium tuberculosis (M. tb) and Mycobacterium bovis (M. bovis) infections holds critical "One Health" significance due to the zoonotic nature of these infections and inherent resistance of M. bovis to pyrazinamide, a key part of the 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 that offers 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 the 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 nanoparticles, which aggregate in the absence of amplified dsDNA on the addition of sodium chloride (NaCl). This technique enables the precise, sensitive, and differential detection of as few as ∼4 bacilli. Our study hence advances tuberculosis detection, overcoming the challenges of M. tb and M. bovis differentiation and offering a quicker alternative to time-consuming methods.