A novel RCA-based DNA sensor system for specific and quantitative detection of Klebsiella pneumonia

Abstract

The timely detection of Klebsiella pneumoniae (K. pneumoniae) infection is of great importance for patient management and the prevention of nosocomial infections. In this study, we present the development of a novel detection assay that enables sensitive and specific identification of K. pneumoniae-encoded topoisomerase IV (K. pneumoniae Topo IV) using a DNA sensor system in combination with Rolling Circle Amplification (RCA) technology. The DNA sensor system was designed by immobilizing DNA hairpin substrates on RCA primers modified slide surface. This modular reacting mode enhances the adaptability and flexibility of the sensor, thereby improving its universality as a sensing platform. When K. pneumoniae is present, the active Topo IV triggers the cleavage-ligation of DNA hairpin substrates, followed by RCA and labeling with fluorescent probes. This results in the visualization of K. pneumoniae at the single-molecule level by the DNA sensor, compared to situations when there are no K. pneumoniae present. The reliability and sensitivity of the DNA sensor were verified using purified enzymes and bacterial extracts, which establishes the foundation for quantitative detection of clinical samples. Notably, the sensor's detection limit for K. pneumoniae extracts was found to be 70 colony-forming units (CFU)/µL, a range considered clinically relevant for infections. Moreover, the DNA sensor system exhibited high sensitivity and accuracy in detecting K. pneumoniae from clinical samples, suggesting its potential application in clinical settings for detecting pathogenic microbes beyond K. pneumoniae.