A smartphone-assisted one-tube ERA-CRISPR/Cas12a biosensor for integrated detection of hypervirulence and antimicrobial resistance determinants in Klebsiella pneumoniae.

The global emergence of hypervirulent Klebsiella pneumoniae (hvKP) strains co-harboring critical antimicrobial resistance determinants pose a serious threat to public health and clinical management. Rapid identification of virulence- and resistance-associated genes is therefore essential for early diagnosis, antimicrobial stewardship, and infection control. In this study, we developed a smartphone-assisted one-tube enzymatic recombinase amplification (ERA)-CRISPR/Cas12a biosensor for simultaneous detection of four hvKP-associated virulence genes (iroB, iucA, peg-344, and rmpA) and two clinically important resistance genes (blaNDM-1 and mcr-1). To overcome the aerosol contamination risk typical of conventional CRISPR diagnostics, our platform features an innovative sealed dual-chamber reaction tube that physically isolates ERA amplification from CRISPR/Cas12a cleavage until manually punctured. The biosensor demonstrates high analytical sensitivity with a limit of detection (LOD) of 1 × 101 CFU/mL and can be fully completed within 45 min under isothermal conditions (42 °C). Furthermore, a custom portable fluorescence detector wirelessly transmits real-time data to a smartphone application for immediate analysis. Importantly, the biosensor exhibited 100% positive agreement with standard qPCR when evaluating blood samples from a mouse infection model and a diverse cohort of 76 clinical K. pneumoniae isolates. Overall, this portable, highly sensitive, and contamination-controlled biosensor provides a robust point-of-care testing (POCT) platform for the rapid screening of multidrug-resistant hvKP.