ORIGINAL RESEARCH article
Front. Cell. Infect. Microbiol.
Sec. Bacteria and Host
Volume 15 - 2025 | doi: 10.3389/fcimb.2025.1653602
This article is part of the Research TopicAdvances in Diagnostic Platforms for Rapid Detection of Multidrug-Resistant Bacterial InfectionsView all articles
Rapid identification of major Mycobacterium species by loop-mediated isothermal amplification assay using novel species-specific genomic targets
Provisionally accepted
- 1Shaanxi Provincial Hospital of Tuberculosis Prevention and Treatment Hospital, Xi’an, China
- 2Xi'an Jiaotong University, Xi'an, China
- 3Shaanxi Provincial People's Hospital, Xi'An, China
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Background: Rapid and precise identification of Mycobacterium species is critical for appropriate clinical management and epidemiological surveillance. However, conventional methods often fail to differentiate closely related nontuberculous mycobacteria (NTM) species, limiting their clinical utility. Methods: We developed a multiplex loop-mediated isothermal amplification (LAMP) assay targeting newly identified species-specific genomic markers for simultaneous detection of Mycobacterium tuberculosis complex (MTBC) and six clinically important NTM species. Analytical performance was assessed using serial dilutions of bacterial cultures and 36 reference strains. Clinical validation was performed on 52 cultured isolates and 349 sputum samples, compared to GeneXpert MTB/RIF and a commercial PCR-reverse dot blot assay. Results: The assay showed high analytical sensitivity, with limits of detection ranging from 76.013 CFU/mL (95% CI: 60.329-113.924 CFU/mL) for MTBC to 166.602-690.629 CFU/mL for NTM species. All reference strains were correctly identified with no cross-reactivity. Among the clinical isolates, all targeted species were accurately detected. One isolate misidentified as M. abscessus by an ITS-based assay was confirmed by sequencing to be M. massiliense, demonstrating the assay's superior discriminatory capacity. For sputum samples, the assay achieved 90.32% sensitivity and 97.55% specificity for MTBC, with an overall agreement of 93.70% (κ = 0.8740). Conclusion: This multiplex LAMP assay offers a rapid, accurate, and field-deployable tool for species-level identification of MTBC and major NTM pathogens. Its simplicity, stability, and compatibility with low-resource settings support its application in routine diagnostics and decentralized tuberculosis programs.
Keywords: Nontuberculous Mycobacteria, Mycobacterium tuberculosis, Loop-mediated isothermal amplification, Species identification, clinical validation
Received: 25 Jun 2025; Accepted: 04 Sep 2025.
Copyright: © 2025 Zou, Wang, Wei, Bai, Wang, Qu, Zhong and Gao. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence:
Guowei Zhong, Shaanxi Provincial Hospital of Tuberculosis Prevention and Treatment Hospital, Xi’an, China
Yanbin Gao, Shaanxi Provincial Hospital of Tuberculosis Prevention and Treatment Hospital, Xi’an, China
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