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Alireza Ekrami1, Ali Reza Samarbaf-Zadeh2, Azar Khosravi1, Behrooz Zargar3, Mohamad Alavi1, Mansor Amin2, Alireza Kiasat3 1Infectious and Tropical Diseases Research Center, Ahvaz Jundishapur University of Medical Sciences, 2Department of Microbiology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, 3Department of Chemistry, School of Science, Shahid Chamran University, Ahvaz, Iran Background: Tuberculosis is a public health problem worldwide, and new easy to perform diagnostic methods with high accuracy are necessary for optimal control of the disease. Recently, fluorescent silica nanoparticles (FSNP) has attracted immense interest for the detection of pathogenic microorganisms. The aim of this study was to detect Mycobacterium tuberculosis in clinical samples using bioconjugated FSNP compared with microscopic examination, polymerase chain reaction (PCR), nested PCR, and culture as the gold standard. Methods: In total, 152 sputum specimens were obtained from patients who were suspected to have pulmonary tuberculosis. All samples were examined by the four techniques described. Results: The assay showed 97.1% sensitivity (95% confidence interval [CI] 91–99.2) and 91.35% specificity (CI 78.3–97.1). Furthermore, assays using variable bacterial concentrations indicated that 100 colony forming units/mL of M. tuberculosis could be detected. There were no differences between the results obtained from two types of mouse monoclonal antibody against Hsp-65 and 16 KDa antigens. Conclusion: We performed this assay in a large number of clinical samples to confirm the diagnostic specificity and sensitivity of the test and can recommend its application for diagnosis of M. tuberculosis. We believe that this method is more convenient for routine diagnosis of M. tuberculosis in sputum and will be more easily applicable in the field, and with sufficient sensitivity. Keywords: Mycobacterium tuberculosis, fluorescent silica nanoparticles, bioconj