Genomic nano-biosensor for rapid detection of carbapenem-resistant gene blaNDM-1 in carbapenemase-producing bacteria

Abstract

Antimicrobial resistance (AMR) has become one of the major public health concerns causing serious obstacles to the successful prevention and treatment of infectious diseases. To curb the spread of AMR, well-equipped laboratories for early detection of disease-causing pathogens and resistant genes are crucial, something that remains unmet in developing countries due to resource constraints and inadequate infrastructure. This paper presents an affordable and simple nanoparticle-based biosensor for rapidly detecting the blaNDM-1 gene in carbapenemase-producing (CP) bacteria. The biosensor employed thiol-ligand surface functionalized gold nanoparticles (GNPs) with conjugated oligonucleotide probe specific to detect the blaNDM-1 gene. The biosensor was evaluated using DNA extracted from CP bacteria having the target blaNDM-1 gene, two non-NDM-1 CP bacteria, and five susceptible bacterial strains. Tuning of the localized surface plasmon resonance (LSPR) of the GNPs was achieved by reducing the surrounding pH of the GNPs, hence inducing aggregation. With the binding of GNPs-probe-target DNA, the stability of GNPs was enhanced and was confirmed by the red colour being maintained when an optimized amount of 0.1 M HCl was added to induce aggregation. The absence of target DNA was indicated by the aggregation of GNPs after the addition of the acid, which corresponded with a colour change from red to blue/purple and shifting the LSPR band to a longer wavelength on average at 620 nm. The biosensor visual detection results were quantified with absorbance spectra measurements and results were achieved within 30 minutes. The biosensor successfully detected the target DNA from blaNDM-1 positive bacteria and distinguished the non-targets. The analytical sensitivity achieved was 2.5 ng/μL which corresponds to approximately 103 colony-forming units per milliliter. These findings were confirmed through PCR amplification. This nano-biosensor advances an inexpensive, simple, rapid, and sensitive method for detecting blaNDM-1 gene in carbapenemase producers, and readily implementable in resource-limited settings.