Selection and application of ssDNA aptamers to detect active TB from sputum samples

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dc.contributor.authorRotherham, Lia Sen_ZA
dc.contributor.authorMaserumule, Charlotteen_ZA
dc.contributor.authorDheda, Keertanen_ZA
dc.contributor.authorTheron, Jacquesen_ZA
dc.contributor.authorKhati, Makobetsaen_ZA
dc.date.accessioned2015-11-23T12:36:52Z
dc.date.available2015-11-23T12:36:52Z
dc.date.issued2012en_ZA
dc.description.abstractBACKGROUND: 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. RESULTS: Twenty-four out of the 66 aptamers that were isolated bound significantly (p<0.05) to the CFP-10.ESAT-6 heterodimer and six were further evaluated. Their dissociation constant (K D ) values were in the nanomolar range. One aptamer, designated CSIR 2.11, was evaluated using sputum samples. CSIR 2.11 had sensitivity and specificity of 100% and 68.75% using Youden's index and 35% and 95%, respectively, using a rule-in cut-point. CONCLUSION: This preliminary proof-of-concept study suggests that a diagnosis of active TB using anti-CFP-10.ESAT-6 aptamers applied to human sputum samples is feasible.en_ZA
dc.identifier.apacitationRotherham, L. S., Maserumule, C., Dheda, K., Theron, J., & Khati, M. (2012). Selection and application of ssDNA aptamers to detect active TB from sputum samples. <i>PLoS One</i>, http://hdl.handle.net/11427/15342en_ZA
dc.identifier.chicagocitationRotherham, Lia S, Charlotte Maserumule, Keertan Dheda, Jacques Theron, and Makobetsa Khati "Selection and application of ssDNA aptamers to detect active TB from sputum samples." <i>PLoS One</i> (2012) http://hdl.handle.net/11427/15342en_ZA
dc.identifier.citationRotherham, L. S., Maserumule, C., Dheda, K., Theron, J., & Khati, M. (2011). Selection and application of ssDNA aptamers to detect active TB from sputum samples. PloS one, 7(10), e46862. doi:10.1371/journal.pone.0046862en_ZA
dc.identifier.ris TY - Journal Article AU - Rotherham, Lia S AU - Maserumule, Charlotte AU - Dheda, Keertan AU - Theron, Jacques AU - Khati, Makobetsa AB - 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. RESULTS: Twenty-four out of the 66 aptamers that were isolated bound significantly (p<0.05) to the CFP-10.ESAT-6 heterodimer and six were further evaluated. Their dissociation constant (K D ) values were in the nanomolar range. One aptamer, designated CSIR 2.11, was evaluated using sputum samples. CSIR 2.11 had sensitivity and specificity of 100% and 68.75% using Youden's index and 35% and 95%, respectively, using a rule-in cut-point. CONCLUSION: This preliminary proof-of-concept study suggests that a diagnosis of active TB using anti-CFP-10.ESAT-6 aptamers applied to human sputum samples is feasible. DA - 2012 DB - OpenUCT DO - 10.1371/journal.pone.0046862 DP - University of Cape Town J1 - PLoS One LK - https://open.uct.ac.za PB - University of Cape Town PY - 2012 T1 - Selection and application of ssDNA aptamers to detect active TB from sputum samples TI - Selection and application of ssDNA aptamers to detect active TB from sputum samples UR - http://hdl.handle.net/11427/15342 ER - en_ZA
dc.identifier.urihttp://hdl.handle.net/11427/15342
dc.identifier.urihttp://dx.doi.org/10.1371/journal.pone.0046862
dc.identifier.vancouvercitationRotherham LS, Maserumule C, Dheda K, Theron J, Khati M. Selection and application of ssDNA aptamers to detect active TB from sputum samples. PLoS One. 2012; http://hdl.handle.net/11427/15342.en_ZA
dc.language.isoengen_ZA
dc.publisherPublic Library of Scienceen_ZA
dc.publisher.departmentDepartment of Medicineen_ZA
dc.publisher.facultyFaculty of Health Sciencesen_ZA
dc.publisher.institutionUniversity of Cape Town
dc.rightsThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.en_ZA
dc.rights.holder© Rotherham et alen_ZA
dc.rights.urihttp://creativecommons.org/licenses/by/4.0en_ZA
dc.sourcePLoS Oneen_ZA
dc.source.urihttp://journals.plos.org/plosoneen_ZA
dc.subject.otherTuberculosisen_ZA
dc.subject.otherSputumen_ZA
dc.subject.otherMycobacterium tuberculosisen_ZA
dc.subject.otherBovine tuberculosisen_ZA
dc.subject.otherMycobacterium bovisen_ZA
dc.subject.otherRecombinant proteinsen_ZA
dc.subject.otherBinding analysisen_ZA
dc.subject.otherTuberculosis diagnosis and managementen_ZA
dc.titleSelection and application of ssDNA aptamers to detect active TB from sputum samplesen_ZA
dc.typeJournal Articleen_ZA
uct.type.filetypeText
uct.type.filetypeImage
uct.type.publicationResearchen_ZA
uct.type.resourceArticleen_ZA
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