A Simple and Label-Free Gold Nanoparticle Assay and Duplex PCR for Rapid Detection of Klebsiella pneumoniae K2

Rapid, accurate, and cost-effective diagnostic tools are crucial for the early detection of bacterial pathogens, particularly Klebsiella pneumoniae, a leading cause of multidrug-resistant infections. In this study, we developed two molecular detection approaches: a simplified, label-free colorimetric assay using unmodified gold nanoparticles (AuNPs) with direct DNA addition, and a duplex PCR assay targeting the K2A and orf10 genes, two key virulence and identification markers of K. pneumoniae. The unmodified AuNP assay, employing a 20 bp K2A gene probe, enabled visible color change detection within 60 min, without the need for complex instrumentation, achieving a sensitivity of 1.56 ng/μL. Duplex PCR successfully amplified fragments of 532 bp (K2A) and 309 bp (orf10), with high sensitivity levels of 1 pg/μL and 0.07 ng/μL, respectively. This integrated strategy offers both visual rapid screening and molecular confirmation in a single workflow, improving diagnostic reliability while maintaining low cost and simplicity. The use of unmodified AuNPs eliminates the need for chemical functionalization, significantly reducing assay preparation time and cost. Together, these features make the proposed method a promising platform for point-of-care molecular diagnostics and epidemiological monitoring of K. pneumoniae in resource-limited settings.