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Abstract <p>This study presents a novel multiplex assay based on capillary electrophoresis (CE) for the simultaneous detection of three <i>Mycobacterium tuberculosis</i> complex (MTBC) genes: <i>IS6110</i>, <i>rpoB</i>, and <i>HSP65</i>. Unlike conventional molecular diagnostic methods that target only a single gene, which may lead to misdiagnosis or missed diagnosis, this CE-based multiplex approach provides comprehensive detection to reduce diagnostic errors. Specificity testing with 76 microorganisms representing common respiratory pathogens confirmed 100% analytical specificity with no cross-reactivity, while sensitivity analysis demonstrated detection limits ranging from 10 to 20 copies/mL for all three target genes. In a prospective clinical validation study of 1067 patients suspected of pulmonary tuberculosis, the multiplex assay showed 77.4% sensitivity (CI 74.9%–79.9%), 99.6% specificity (CI 99.2%–100%), 96.0% positive predictive value (CI 94.8%–97.2%), and 97.1% negative predictive value (CI 96.1%–98.1%). Notably, the study identified 6 MTBC strains (4.8% of TB patients) with <i>IS6110</i> deletions through whole-genome sequencing, which would result in false-negative results for any commercial PCR kits targeting <i>IS6110</i>. This integrated multiplex approach enhances diagnostic accuracy by simultaneously targeting multiple genes; then it offers the potential to reduce misdiagnosis and missed diagnosis of tuberculosis. In summary, the multiplex assay provides a more comprehensive alternative to current single-target molecular methods for MTBC detection.</p> Key points <p>• <i>The multiplex assay provides one-run results for IS6110, rpoB, and HSP65.</i></p> <p>•&#xa0;<i>The multiplex assay is a more comprehensive method to detect MTBC.</i></p> <p>•&#xa0;<i>This approach can reduce misdiagnosis and missed diagnosis of TB.</i></p>

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A multiplex assay based on capillary electrophoresis to detect Mycobacterium tuberculosis complex: development and clinical validation

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年2月2日

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  • Yaocheng Wang,
  • Zhen Li,
  • Li Lai,
  • Yiping Liu,
  • Li Li,
  • Yi Huang

摘要

Abstract

This study presents a novel multiplex assay based on capillary electrophoresis (CE) for the simultaneous detection of three Mycobacterium tuberculosis complex (MTBC) genes: IS6110, rpoB, and HSP65. Unlike conventional molecular diagnostic methods that target only a single gene, which may lead to misdiagnosis or missed diagnosis, this CE-based multiplex approach provides comprehensive detection to reduce diagnostic errors. Specificity testing with 76 microorganisms representing common respiratory pathogens confirmed 100% analytical specificity with no cross-reactivity, while sensitivity analysis demonstrated detection limits ranging from 10 to 20 copies/mL for all three target genes. In a prospective clinical validation study of 1067 patients suspected of pulmonary tuberculosis, the multiplex assay showed 77.4% sensitivity (CI 74.9%–79.9%), 99.6% specificity (CI 99.2%–100%), 96.0% positive predictive value (CI 94.8%–97.2%), and 97.1% negative predictive value (CI 96.1%–98.1%). Notably, the study identified 6 MTBC strains (4.8% of TB patients) with IS6110 deletions through whole-genome sequencing, which would result in false-negative results for any commercial PCR kits targeting IS6110. This integrated multiplex approach enhances diagnostic accuracy by simultaneously targeting multiple genes; then it offers the potential to reduce misdiagnosis and missed diagnosis of tuberculosis. In summary, the multiplex assay provides a more comprehensive alternative to current single-target molecular methods for MTBC detection.

Key points

• The multiplex assay provides one-run results for IS6110, rpoB, and HSP65.

• The multiplex assay is a more comprehensive method to detect MTBC.

• This approach can reduce misdiagnosis and missed diagnosis of TB.