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

## TL;DR

A new test using capillary electrophoresis detects three tuberculosis-related genes at once, improving diagnostic accuracy and reducing errors.

## Contribution

A novel multiplex capillary electrophoresis assay for simultaneous detection of three MTBC genes, enhancing diagnostic accuracy.

## Key findings

- The assay detected MTBC genes IS6110, rpoB, and HSP65 with 100% analytical specificity and no cross-reactivity.
- Clinical validation showed 77.4% sensitivity and 99.6% specificity in diagnosing tuberculosis.
- Six MTBC strains with IS6110 deletions were identified, which would be missed by single-gene PCR tests.

## 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.

• 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.

The online version contains supplementary material available at 10.1007/s00253-025-13701-0.