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<p>Conventional susceptibility testing requires 18–48&#xa0;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 (<i>bla</i><sub>KPC</sub>, <i>bla</i><sub>NDM</sub>, <i>bla</i><sub>OXA-48-like</sub>, <i>bla</i><sub>VIM</sub>, <i>bla</i><sub>IMP</sub>, <i>mcr-1</i>, and <i>mcr-3</i>). 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 &lt; 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 <i>bla</i><sub>NDM</sub> and <i>bla</i><sub>OXA-48-like</sub> 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% (R<sup>2</sup> = 0.96–0.99), and a minimum of 10<sup>2</sup> copies required to confidently detect all targets. In under 4&#xa0;h, this cost-effective assay leverages existing platforms for comprehensive surveillance of carbapenem and colistin resistance, enabling timely antimicrobial stewardship and infection control.</p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年3月3日

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  • Sirirat Luk-In,
  • Kamonrat Phopin,
  • Sasina Bangmuangngam,
  • Tanittha Chatsuwan,
  • Dhammika Leshan Wannigama,
  • Aye Mya Sithu Shein,
  • Ratana Lawung,
  • Tanawut Tantimongcolwat

摘要

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 (blaKPC, blaNDM, blaOXA-48-like, blaVIM, blaIMP, 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 blaNDM and blaOXA-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.