Reza Kamali Kakhki1, Alireza Neshani2, Mojtaba Sankian3, Kiarash Ghazvini1, Amin Hooshyar1, Mahsa Sayadi1. 1. Antimicrobial Resistance Research Center, Mashhad University of Medical Sciences, Mashhad, Iran. 2. Antimicrobial Resistance Research Center, Mashhad University of Medical Sciences, Mashhad, Iran; Student Research Committee, Mashhad University of Medical Sciences, Mashhad, Iran. 3. Immunology Research Center, Bu-Ali Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran. Electronic address: sankianm@mums.ac.ir.
Abstract
BACKGROUND: Early detection of tuberculosis is one of the crucial steps for TB control. Although, the sensitivity of conventional methods like Lowenstein Jensen (LJ) culture and direct staining is quite low, molecular techniques like polymerase chain reaction (PCR) are more sensitive and be considered as useful tools for rapid detection of tuberculosis. Various genes like IS6110 and mpb64 have been used as target for detection of M. tuberculosis, but more research is needed to find the most specific targets. The short-chain dehydrogenases/reductases family (SDR) is one of a very large family of NAD- or NADP-dependent oxidoreductase enzymes which is present in all M. tuberculosis strains. The large part of SDR sequences in tuberculosis is completely conserved and different from non-tuberculosis mycobacterium. The aim of the study was to develop an in-house PCR assay using the SDR target for rapid detection of M. tuberculosis from clinical specimens. METHOD: M. tuberculosis-specific sequences were found using modified genome comparison method and the primers were designed by the Primer Premier 5.0 software. A PCR assay was developed targeting the nucleotide sequences within the SDR gene. A total of 50 cultivated specimens and 120 clinical specimens were evaluated by PCR. RESULTS: The clinical evaluation of SDR PCR assay showed high specificity (100%) and high sensitivity (88.5%). The analytical sensitivity was 10 fg of template DNA which is theoretically equivalent to 2 copy of genomic DNA per microliter. The SDR is a new specific target of M. tuberculosis and no cross-reactivity was observed to non-tuberculosis mycobacterium and other pathogenic bacteria. CONCLUSIONS: Based on our results, the SDR gene can be considered as a useful target for detection of M. tuberculosis complex from clinical specimens.
BACKGROUND: Early detection of tuberculosis is one of the crucial steps for TB control. Although, the sensitivity of conventional methods like Lowenstein Jensen (LJ) culture and direct staining is quite low, molecular techniques like polymerase chain reaction (PCR) are more sensitive and be considered as useful tools for rapid detection of tuberculosis. Various genes like IS6110 and mpb64 have been used as target for detection of M. tuberculosis, but more research is needed to find the most specific targets. The short-chain dehydrogenases/reductases family (SDR) is one of a very large family of NAD- or NADP-dependent oxidoreductase enzymes which is present in all M. tuberculosis strains. The large part of SDR sequences in tuberculosis is completely conserved and different from non-tuberculosismycobacterium. The aim of the study was to develop an in-house PCR assay using the SDR target for rapid detection of M. tuberculosis from clinical specimens. METHOD:M. tuberculosis-specific sequences were found using modified genome comparison method and the primers were designed by the Primer Premier 5.0 software. A PCR assay was developed targeting the nucleotide sequences within the SDR gene. A total of 50 cultivated specimens and 120 clinical specimens were evaluated by PCR. RESULTS: The clinical evaluation of SDR PCR assay showed high specificity (100%) and high sensitivity (88.5%). The analytical sensitivity was 10 fg of template DNA which is theoretically equivalent to 2 copy of genomic DNA per microliter. The SDR is a new specific target of M. tuberculosis and no cross-reactivity was observed to non-tuberculosismycobacterium and other pathogenic bacteria. CONCLUSIONS: Based on our results, the SDR gene can be considered as a useful target for detection of M. tuberculosis complex from clinical specimens.