AUTHOR=Zou Yuanwu , Wang Zhuo , Wei Zihan , Bai Guanghong , Wang Xiaolin , Qu Shaoyi , Zhong Guowei , Gao Yanbin 

TITLE=Rapid identification of major Mycobacterium species by loop-mediated isothermal amplification assay using novel species-specific genomic targets

JOURNAL=Frontiers in Cellular and Infection Microbiology

VOLUME=Volume 15 - 2025

YEAR=2025

URL=https://www.frontiersin.org/journals/cellular-and-infection-microbiology/articles/10.3389/fcimb.2025.1653602

DOI=10.3389/fcimb.2025.1653602

ISSN=2235-2988

ABSTRACT=BackgroundRapid 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.MethodsWe 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.ResultsThe 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).ConclusionThis 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.