caa a22 4500 605505691 CHVBK 20210128100623.0 cr unu---uuuuu 210128e20151101xx s 000 0 eng 10.1007/s00253-015-6815-7 doi (NATIONALLICENCE)springer-10.1007/s00253-015-6815-7 Identification and application of ssDNA aptamers against H37Rv in the detection of Mycobacterium tuberculosis [Elektronische Daten] [Rusitanmujiang Aimaiti, Lianhua Qin, Ting Cao, Hua Yang, Jie Wang, Junmei Lu, Xiaochen Huang, Zhongyi Hu] Microscopy of direct smear with the Ziehl-Neelsen stain is still broadly used in tuberculosis diagnosis. However, this method suffers from low specificity and is difficult to distinguish Mycobacterium tuberculosis (MTB) from nontuberculosis mycobacterial (NTM), since all mycobacterial species are positive in Ziehl-Neelsen stain. In this study, we utilized whole cell SELEX to obtain species-specific aptamers for increasing the specificity of MTB detection. Whole cell SELEX was performed in MTB reference strain H37Rv by two selection processes based on enzyme-linked plate or Eppendorf tube, respectively. To increase success rate of generating aptamers, the selection processes were systematically monitored to understand the dynamic evolution of aptamers against complex structure of target bacteria. Two preponderant groups and ten high-affinity aptamers were obtained by analyzing the dynamic evolution. Preponderant aptamer MA1 from group I showed relatively high binding affinity with apparent dissociation constant (KD value) of 12.02 nM. Sandwich ELISA assay revealed five aptamer combinations effectively bound MTB strains in preliminary evaluation, especially the combination based on aptamer MA2 (another preponderant aptamer from group II) and MA1. Further evaluated in many other strains, MA2/MA1 combination effectively identified MTB from NTM or other pathogenic bacteria, and displayed the high specificity and sensitivity. Binding analysis of aptamer MA1 or MA2 by fluorescence microscopy observation showed high binding reactivity with H37Rv, low apparent cross-reactivity with M. marinum, and no apparent cross-reactivity with Enterobacter cloacae. Taken together, this study provides attractive candidate species-specific aptamers to effectively capture or discriminate MTB strains. Springer-Verlag Berlin Heidelberg, 2015 Evolution nationallicence Whole cell SELEX nationallicence ssDNA aptamer nationallicence Mycobacterium tuberculosis nationallicence Detection nationallicence Aimaiti Rusitanmujiang Shanghai Key Laboratory of Tuberculosis, Clinic and Research Center of Tuberculosis, Shanghai Pulmonary Hospital, Tongji University School of Medicine, 507 Zhengmin Road, 200433, Shanghai, China aut Qin Lianhua Shanghai Key Laboratory of Tuberculosis, Clinic and Research Center of Tuberculosis, Shanghai Pulmonary Hospital, Tongji University School of Medicine, 507 Zhengmin Road, 200433, Shanghai, China aut Cao Ting Shanghai Key Laboratory of Tuberculosis, Clinic and Research Center of Tuberculosis, Shanghai Pulmonary Hospital, Tongji University School of Medicine, 507 Zhengmin Road, 200433, Shanghai, China aut Yang Hua Shanghai Key Laboratory of Tuberculosis, Clinic and Research Center of Tuberculosis, Shanghai Pulmonary Hospital, Tongji University School of Medicine, 507 Zhengmin Road, 200433, Shanghai, China aut Wang Jie Shanghai Key Laboratory of Tuberculosis, Clinic and Research Center of Tuberculosis, Shanghai Pulmonary Hospital, Tongji University School of Medicine, 507 Zhengmin Road, 200433, Shanghai, China aut Lu Junmei Shanghai Key Laboratory of Tuberculosis, Clinic and Research Center of Tuberculosis, Shanghai Pulmonary Hospital, Tongji University School of Medicine, 507 Zhengmin Road, 200433, Shanghai, China aut Huang Xiaochen Shanghai Key Laboratory of Tuberculosis, Clinic and Research Center of Tuberculosis, Shanghai Pulmonary Hospital, Tongji University School of Medicine, 507 Zhengmin Road, 200433, Shanghai, China aut Hu Zhongyi Shanghai Key Laboratory of Tuberculosis, Clinic and Research Center of Tuberculosis, Shanghai Pulmonary Hospital, Tongji University School of Medicine, 507 Zhengmin Road, 200433, Shanghai, China aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/21(2015-11-01), 9073-9083 0175-7598 99:21<9073 2015 99 253 https://doi.org/10.1007/s00253-015-6815-7 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-015-6815-7 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Aimaiti Rusitanmujiang Shanghai Key Laboratory of Tuberculosis, Clinic and Research Center of Tuberculosis, Shanghai Pulmonary Hospital, Tongji University School of Medicine, 507 Zhengmin Road, 200433, Shanghai, China aut NATIONALLICENCE 700 1- Qin Lianhua Shanghai Key Laboratory of Tuberculosis, Clinic and Research Center of Tuberculosis, Shanghai Pulmonary Hospital, Tongji University School of Medicine, 507 Zhengmin Road, 200433, Shanghai, China aut NATIONALLICENCE 700 1- Cao Ting Shanghai Key Laboratory of Tuberculosis, Clinic and Research Center of Tuberculosis, Shanghai Pulmonary Hospital, Tongji University School of Medicine, 507 Zhengmin Road, 200433, Shanghai, China aut NATIONALLICENCE 700 1- Yang Hua Shanghai Key Laboratory of Tuberculosis, Clinic and Research Center of Tuberculosis, Shanghai Pulmonary Hospital, Tongji University School of Medicine, 507 Zhengmin Road, 200433, Shanghai, China aut NATIONALLICENCE 700 1- Wang Jie Shanghai Key Laboratory of Tuberculosis, Clinic and Research Center of Tuberculosis, Shanghai Pulmonary Hospital, Tongji University School of Medicine, 507 Zhengmin Road, 200433, Shanghai, China aut NATIONALLICENCE 700 1- Lu Junmei Shanghai Key Laboratory of Tuberculosis, Clinic and Research Center of Tuberculosis, Shanghai Pulmonary Hospital, Tongji University School of Medicine, 507 Zhengmin Road, 200433, Shanghai, China aut NATIONALLICENCE 700 1- Huang Xiaochen Shanghai Key Laboratory of Tuberculosis, Clinic and Research Center of Tuberculosis, Shanghai Pulmonary Hospital, Tongji University School of Medicine, 507 Zhengmin Road, 200433, Shanghai, China aut NATIONALLICENCE 700 1- Hu Zhongyi Shanghai Key Laboratory of Tuberculosis, Clinic and Research Center of Tuberculosis, Shanghai Pulmonary Hospital, Tongji University School of Medicine, 507 Zhengmin Road, 200433, Shanghai, China aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/21(2015-11-01), 9073-9083 0175-7598 99:21<9073 2015 99 253