selection and application of ssdna aptamers to detect active tb from sputum samples选择和应用ssdna寡核苷酸适配子从痰标本检测活动性结核病.pdfVIP

Selection and Application of ssDNA Aptamers to Detect Active TB from Sputum Samples 1,2 1,3 3 2 1,3 Lia S. Rotherham , Charlotte Maserumule , Keertan Dheda , Jacques Theron , Makobetsa Khati * 1 Emerging Health Technologies Platform, Council for Scientific and Industrial Research, Biosciences Unit, Pretoria, South Africa, 2 Department of Microbiology and Plant Pathology, University of Pretoria, Pretoria, South Africa, 3 Department of Medicine, Groote Schuur Hospital and University of Cape Town, Cape Town, South Africa Abstract Background: Despite the enormous global burden of tuberculosis (TB), conventional approaches to diagnosis continue to rely on tests that have major drawbacks. The improvement of TB diagnostics relies, not only on good biomarkers, but also upon accurate detection methodologies. The 10-kDa culture filtrate protein (CFP-10) and the 6-kDa early secreted antigen target (ESAT-6) are potent T-cell antigens that are recognised by over 70% of TB patients. Aptamers, a novel sensitive and specific class of detection molecules, has hitherto, not been raised to these relatively TB-specific antigens. Methods: DNA aptamers that bind to the CFP-10.ESAT-6 heterodimer were isolated. To assess their affinity and specificity to the heterodimer, aptamers were screened using an enzyme-linked oligonucleotide assay (ELONA). One suitable aptamer was evaluated by ELONA using sputum samples obtained from 20 TB patients and 48 control patients (those with latent TB infection, symptomatic non TB patients, and healthy laboratory volunteers). Culture positivity for Mycobacterium tuberculosis (Mtb) served as the reference standard. Accuracy and cut-points were evaluated using ROC curve analysis.