A rapid detection system for core virulence and resistance genes in hypervirulent Klebsiella pneumoniae using multiplex fluorescence PCR-capillary electrophoresis

Infections caused by hypervirulent Klebsiella pneumoniae (hvKp) have increased in clinical settings, yet rapid methods for integrated virulence–serotype–resistance profiling remain underdeveloped. In this study, we developed a multiplex fluorescence PCR-capillary electrophoresis (MPCE) system for the rapid and simultaneous detection of core genetic markers in hvKp. Specifically, the MPCE assay detected 16 genetic markers: five virulence genes ( iucA , iroB , peg344 , rmpA , and rmpA2 ), two major capsular serotype genes (K1 and K2), seven resistance genes ( bla KPC , blaNDM, bla CTX-M , bla SHV , bla OXA-23 , bla OXA-48 and mcr-1 ), and two internal controls. Amplicons were subsequently analyzed via capillary electrophoresis and GeneMapper software. As a result, the MPCE system simultaneously detected all 16 targets in 152 min, demonstrating single-base resolution that enabled precise discrimination between closely related amplicons. Moreover, the assay achieved a limit of detection of 10 2 copies/μL, exhibited excellent repeatability, and showed no cross-reactivity against a panel of non-target pathogens. Furthermore, clinical validation confirmed its strong concordance with next-generation sequencing ( κ = 0.659–1.000). Therefore, the MPCE-based assay provides a high-throughput, sensitive, and specific platform that enables simultaneous profiling of virulence and resistance genes for the comprehensive genotyping of hvKp. It represents a valuable tool for enhancing antimicrobial resistance surveillance and epidemiological investigations.