en <p><span style="font-size:11pt">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.</span></p> <p><span style="font-size:11pt">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. </span></p> <p><span style="font-size:11pt">Results: The MST601-PCR assay demonstrated high analytical sensitivity, with a limit of detection of ~10 fg (&asymp;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 &ge;99% identity to reference M. simiae strains in the GenBank database, validating the assay&#39;s accuracy.</span></p> <p><span style="font-size:11pt">Conclusion: A species-specific nucleic acid target, MST601, facilitating the rapid and accurate detection of M. simiae via conventional PCR was presented. This assay pro-vides a low-cost and accessible option for diagnostic laboratories.</span></p> Molecular diagnostics, MST601, Mycobacterium simiae, Novel target, PCR, Sensitivity, Specificity 157 166 https://www.ajmb.org/En/Article.aspx?id=70652 https://www.ajmb.org/PDF/En/FullText/70652.pdf RezaKamali KakhkiMashhad Gene Azma Inc., Mashhad, Iran91889MohammadAbavisani Student Research Committee, Mashhad University of Medical Sciences, Mashhad, Iran92504KiarashGhazvini Department of Microbiology and Virology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran92505HosnaZareDepartment of Laboratory Sciences, School of Paramedical Sciences, Mashhad University of Medical Sciences, Mashhad, Iran91888NoshinHojatpanah Department of Microbiology and Virology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran92507JamalFalahi Department of Laboratory Sciences, School of Paramedical Sciences, Torbat Heydariyeh University of Medical Sciences, Torbat Heydariyeh, Iran92508AlirezaNeshaniStudent Research Committee, Mashhad University of Medical Sciences, Mashhad, Iran31529