title: Identification and application of ssDNA aptamers against H₃₇Rv in the detection of Mycobacterium tuberculosis.
abstract: 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.
authors.affiliation: Shanghai Key Laboratory of Tuberculosis, Clinic and Research Center of Tuberculosis, Shanghai Pulmonary Hospital, Tongji University School of Medicine, 507 Zhengmin Road, Shanghai, 200433, China.
authors.forename: Rusitanmujiang
authors.initials: R
authors.lastname: Aimaiti
authors.affiliation: Shanghai Key Laboratory of Tuberculosis, Clinic and Research Center of Tuberculosis, Shanghai Pulmonary Hospital, Tongji University School of Medicine, 507 Zhengmin Road, Shanghai, 200433, China.
authors.forename: Lianhua
authors.initials: L
authors.lastname: Qin
authors.affiliation: Shanghai Key Laboratory of Tuberculosis, Clinic and Research Center of Tuberculosis, Shanghai Pulmonary Hospital, Tongji University School of Medicine, 507 Zhengmin Road, Shanghai, 200433, China.
authors.forename: Ting
authors.initials: T
authors.lastname: Cao
authors.affiliation: Shanghai Key Laboratory of Tuberculosis, Clinic and Research Center of Tuberculosis, Shanghai Pulmonary Hospital, Tongji University School of Medicine, 507 Zhengmin Road, Shanghai, 200433, China.
authors.forename: Hua
authors.initials: H
authors.lastname: Yang
authors.affiliation: Shanghai Key Laboratory of Tuberculosis, Clinic and Research Center of Tuberculosis, Shanghai Pulmonary Hospital, Tongji University School of Medicine, 507 Zhengmin Road, Shanghai, 200433, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Wang
authors.affiliation: Shanghai Key Laboratory of Tuberculosis, Clinic and Research Center of Tuberculosis, Shanghai Pulmonary Hospital, Tongji University School of Medicine, 507 Zhengmin Road, Shanghai, 200433, China.
authors.forename: Junmei
authors.initials: J
authors.lastname: Lu
authors.affiliation: Shanghai Key Laboratory of Tuberculosis, Clinic and Research Center of Tuberculosis, Shanghai Pulmonary Hospital, Tongji University School of Medicine, 507 Zhengmin Road, Shanghai, 200433, China.
authors.forename: Xiaochen
authors.initials: X
authors.lastname: Huang
authors.affiliation: Shanghai Key Laboratory of Tuberculosis, Clinic and Research Center of Tuberculosis, Shanghai Pulmonary Hospital, Tongji University School of Medicine, 507 Zhengmin Road, Shanghai, 200433, China. shhuzy@163.com.
authors.forename: Zhongyi
authors.initials: Z
authors.lastname: Hu
mesh_terms: D052157:Aptamers, Nucleotide / Q000235:genetics / Q000378:metabolism*; D001431:Bacteriological Techniques / Q000379:methods*; D003429:Cross Reactions; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000302:isolation & purification*; D052156:SELEX Aptamer Technique; D012680:Sensitivity and Specificity; D014376:Tuberculosis / Q000175:diagnosis*
chemical_list: D052157:Aptamers, Nucleotide
keywords: Detection; Evolution; Mycobacterium tuberculosis; Whole cell SELEX; ssDNA aptamer