Multiplex real-time PCR with high-resolution melting analysis for rapid identification of carbapenem and colistin resistance genes in clinical Enterobacterales isolates

Conventional susceptibility testing requires 18-48 h and often delays therapy, whereas existing molecular assays are costly and limited. Real-time PCR's ubiquity in hospitals offers a rapid multiplex screening platform. We established a single-tube multiplex real-time PCR assay coupled with high-resolution melting analysis to detect seven resistance genes (bla_KPC, bla_NDM, bla_OXA-48-like, bla_VIM, bla_IMP, mcr-1, and mcr-3). Validated on 577 clinical Enterobacterales isolates and a standard strain, the assay exhibited distinct melt peaks for each target, with intra- and inter-run CVs < 0.1%. Compared with reference PCR, the assay offered overall sensitivity of 97.3%, the specificity of 99.5%, the PPV of 99.7%, and the NPV of 95.4%, yielding a kappa coefficient of 0.936 (95% CI 0.913-0.958) with "high concordance". Codetection of the bla_NDM and bla_OXA-48-like genes improved the sensitivity from 82.7% to 92.9% when precision melt analysis software was used. The assay demonstrated good quantitative analytical performance, with efficiencies ranging from 91 to 114% (R² = 0.96-0.99), and a minimum of 10² copies required to confidently detect all targets. In under 4 h, this cost-effective assay leverages existing platforms for comprehensive surveillance of carbapenem and colistin resistance, enabling timely antimicrobial stewardship and infection control.