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

Abstract

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.

Unique combination of Recombinase Polymerase Amplification with label-free citrate-stabilised silver nanoparticle assay offers a highly sensitive, fast, accurate and visual molecular detection ofKlebsiella pneumoniae

“…The RPA technique for isothermal DNA amplification, distinguishes itself from PCR by the virtue of its speed of operation (15-20 min) at a constant temperature range (∼25 - 42°C) compared to other isothermal techniques like LAMP and MCDA requiring more time (∼30 – 60 min) and higher temperature (60 - 65 °C). AgNP-based assay reported by us previously for differential molecular detection of Mycobacterium tuberculosis and M. bovis 29 , offers unique advantage of visual detection in a highly sensitive and cost-effective manner. The synergy of RPA and the AgNP assay eliminates the need for expensive thermal cycling equipment, centrifuges, electrophoresis equipment, gel documentation facilities, and real-time qPCR machines, making RPA particularly suitable for field and POC applications.…”

“…Bridging flocculation assay and AgNP-based visual detection assays were performed using PCR amplified products as described by us previously 29 . Briefly, paramagnetic beads work with a principle that they adsorb the amplified DNA molecules and flocculate upon addition of flocculation buffer (sodium acetate and tween 20 at an acidic pH) due to the formation of DNA bridges connecting the paramagnetic beads.…”

“…Absence of DNA or lesser amount of the amplified product doesn’t form visible floc as DNA bridges are not formed, as indicated by brown dispersion 29 . Similarly, AgNP assay was carried out as described by us previously 29 , wherein presence of DNA prevents the aggregation of AgNPs upon addition of aggregating agent, sodium chloride (NaCl) causing sliver dispersion to maintain yellow colour. However, in absence of DNA, addition of NaCl leads to AgNP aggregation causing grey coloration of the dispersion.…”

“…This study combines three unique POC friendly methods (a) InstaDNA card, for easy sample collection and DNA extraction (b) RPA-based thermal amplification in addition to PCR (c) application of silver nanoparticle (AgNP) aggregation assay developed in our laboratory 29 for rapid, sensitive and visual molecular detection of K. pneumoniae . This unique combination is particularly suitable for K. pneumoniae detection in resource-poor settings without the need of costly thermal cycler and equipment like centrifuge or qPCRs.…”