Identification of a Novel Nucleic Acid Target for the Rapid and Specific Detection  of Mycobacterium Simiae Using Comparative Genomic Analysis

Reza Kamali  Kakhki; Mohammad   Abavisani; Kiarash  Ghazvini; Hosna  Zare; Noshin  Hojatpanah; Jamal  Falahi; Alireza  Neshani

doi:10.18502/ajmb.v18i2.21508

Reza Kamali Kakhki Mashhad Gene Azma Inc., Mashhad, Iran
Mohammad Abavisani Student Research Committee, Mashhad University of Medical Sciences, Mashhad, Iran
Kiarash Ghazvini Department of Microbiology and Virology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
Hosna Zare Student Research Committee, Mashhad University of Medical Sciences, Mashhad, Iran
Noshin Hojatpanah Student Research Committee, Mashhad University of Medical Sciences, Mashhad, Iran
Jamal Falahi Department of Laboratory Sciences, School of Paramedical Sciences, Torbat Heydariyeh University of Medical Sciences, Torbat Heydariyeh, Iran
Alireza Neshani Student Research Committee, Mashhad University of Medical Sciences, Mashhad, Iran

DOI: https://doi.org/10.18502/ajmb.v18i2.21508

Keywords: Molecular diagnostics, MST601, Mycobacterium simiae, Novel target, PCR, Sensitivity, Specificity

Abstract

Background: Mycobacterium simiae (M. simiae) is a non-tuberculous mycobacterium (NTM) that closely resembles Mycobacterium tuberculosis (M. tuberculosis) in clinical and biochemical characteristics, notably its niacin-positive phenotype. This similarity frequently leads to misdiagnosis and inappropriate treatment with first-line anti-tuberculosis drugs, to which M. simiae is frequently resistant. Current diagnostic methods are expensive or need complex equipment, highlighting the urgent need for a rapid, specific, and accessible molecular target to identify M. simiae accurately.

Methods: In this study, a modified genome comparison method was applied to the complete reference genome of M. simiae (AP022568.1) in order to identify a putative species-specific nucleotide sequence. A conventional PCR assay was designed to amplify a 168-bp fragment within this target, designated MST601 (M. simiae Target, 601 bp). The analytical sensitivity [Limit of Detection (LOD)] was determined using serial dilutions of genomic DNA. The pilot evaluation of the assay was assessed using 10 well-characterized clinical isolates of M. simiae.

Results: The MST601-PCR assay demonstrated high analytical sensitivity, with a limit of detection of ~10 fg (≈2 genome equivalents per reaction) of M. simiae genomic DNA. No cross-reactivity among the tested species was observed with any of the 10 non-target mycobacterial species tested. The assay successfully amplified the target sequence from all 10 clinical isolates. Sequencing of the amplicons revealed ≥99% identity to reference M. simiae strains in the GenBank database, validating the assay's accuracy.

Conclusion: A species-specific nucleic acid target, MST601, facilitating the rapid and accurate detection of M. simiae via conventional PCR was presented. This assay provides a low-cost and accessible option for diagnostic laboratories.