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

Our study addresses the growing concern posed by Klebsiella pneumoniae, a significant pathogen acknowledged by the World Health Organization (WHO). This bacterium is particularly alarming due to its association with antimicrobial resistance (AMR), impacting immunologically vulnerable populations, especially in hospital settings, and playing a crucial role in wound management. Moreover, this pathogen raises significant concerns in maternal and child health, being correlated with adverse outcomes like pre-term birth, low birth weight, and increased susceptibility to infections in new-borns, often resulting in morbidity and mortality. A major obstacle to the effective and timely management of K. pneumoniae infections is the absence of rapid and cost-effective detection tools in resource-poor point-of-care (POC) settings. This study introduces an innovative combination of three POC-compatible methods: Insta DNA card-based sample collection and DNA extraction, Recombinase polymerase amplification (RPA)-based isothermal amplification, and a silver nanoparticle (AgNP) aggregation assay for visual detection. Together, these methods offer simple yet highly sensitive, specific, and rapid visual detection of as few as [~]1 bacterium of K. pneumoniae within [~]45 minutes. The synergy of these methods eliminates the need for sophisticated equipment, making it highly suitable for field and resource-poor POC applications.