Alpha/Beta Hydrolase Gene as a Novel Specific Target for <i>Mycobacterium simiae</i> Using Computational Analyses

Abstract

Background: Mycobacterium simiae, a slow-growing nontuberculous mycobacterium (NTM), poses diagnostic and therapeutic challenges because of its clinical similarity to Mycobacterium tuberculosis and its resistance to standard antituberculosis drugs. Accurate and rapid identification is critical, particularly in immunocompromised individuals. Therefore, species-specific diagnostic targets are essential for differentiating among Mycobacterium species. Objectives: To identify and evaluate a novel species-specific genetic target for the detection of Mycobacterium simiae. Methods: An innovative approach was used to identify a unique and conserved genetic target specific to M. simiae. Two complete genomes were evaluated in silico using a modified genome-comparison approach to detect conserved coding sequences. A specific gene, G6N33_RS00430, encoding an alpha/beta hydrolase (ABH), was selected. Specific primers were designed, assessed for secondary structures, and screened against human and mycobacterial genomes to confirm their specificity. The ABH-PCR assay was validated using genomic DNA from various mycobacterial species and clinical isolates. Results: The ABH gene was confirmed to be unique to M. simiae, with minimal similarity to closely related species. The PCR assay demonstrated high analytical specificity, with amplification observed exclusively in M. simiae. Sensitivity testing indicated a detection limit of 5 fg of genomic DNA. All 10 clinical isolates yielded positive results, and 5 were confirmed by sequencing. Conclusions: This study identifies a novel species-specific genetic target for M. simiae, enabling rapid, accurate, and cost-effective PCR-based identification. The ABH-PCR assay shows strong potential for routine diagnostic use, particularly in resource-limited settings, and helps address a critical gap in NTM diagnostics.