Selection and application of ssDNA aptamers to detect active TB from sputum samples
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This document presents a study on the selection and application of single-stranded DNA (ssDNA) aptamers for detecting active tuberculosis (TB) from sputum samples. The research discusses the use of aptamers targeting the esat-6/cfp-10 complex, their affinity, and specificity, achieving a high sensitivity of 100% and a specificity of 68.75%, indicating promising potential for TB diagnosis. Future work includes optimizing aptamers and validating results with larger clinical samples.
Selection and application of ssDNA aptamers to detect active TB from sputum samples
- 1. Selection and application of ssDNA aptamer to detect active TB from sputum samples Date: /4/14 Presented by Toh Saw Yi Impact factor: 3.73 5-year IF: 4.24
- 2. The Tuberculosis Threat Tuberculosis (TB) facts: Approximately 8.6 million new TB cases and 1.3 million deaths in 2012 (World Health Organization, 2013) Spreads via airborne droplets (Mayo Foundation for Medical Education and Research (MFMER), 2011) infectees - rising global human population; rising number of people with immune-deficient diseases (World Health Organization, 2010) World Health Organization (WHO) - Stop TB strategy – aims to reduce the global threat of TB by 2015 (Hawthorne, Thickett, & Wood, 2012).
- 3. What is ESAT-6? ESAT-6 6kDa Early secretory protein Specific to virulent members of the MTB complex Potent T-cell antigen – induces interferon γ Functions: Virulence of MTB (Renshaw et al., 2005) Phagosomal rupture of phagocytes (Simeone et al., 2012) Inhibits downstream signal transduction in macrophages (S. K. Pathak et al., 2007) And others…
- 4. What is CFP-10? CFP-10 ESAT-6-like protein esxB/ secreted antigenic protein MTSA-10/ 10 kDa culture filtrate antigen Forms a tight 1:1 heterodimeric complex with ESAT-6 Functions: Virulence of MTB (Renshaw et al., 2005)
- 5. What is ESAT-6/CFP-10 complex? Complex 100AA sequence Has a hydrophobic nature Has high content of α-helical structures Are recognised in over 70% of TB patients Functions: Virulence of MTB (Renshaw et al., 2005)
- 6. Why DNA aptamer? • Reportedly, they state that DNA aptamers: 1. Possess greater inherent stability (DNA) 2. Are cheaper to produce than RNA aptamer
- 7. What they have done? Their aptamer… • 90-mer DNA aptamer with 49 randomised sequence How to get ssDNA from dsDNA after PCR? • 2 methods: T7-based SELEX and exonuclease-based SELEX dsDNA used as a template to obtain RNA (via in vitro transcription) -> +DNase I, purified and preciptated -> RT-PCR -> Hydrolyse RNA -> purify cDNA -> NEXT round of SELEX Digestion of dsDNA with lambda exonuclease -> degradation of the reverse strand -> NEXT round of SELEX • Negative selection: Pool is subjected to counter-selection against nitrocellulose partitioning membrane Known region Known regionRandom Nt region (49 bases)
- 8. Comparison between T7-based SELEX and exonuclease-based SELEX • Exonuclease-based SELEX has an enrichment of 68% after 4 rounds of SELEX. • T7-based SELEX requires more rounds to hit 54.7% enrichment
- 9. • Check binding for final enriched pool of aptamer via ELONA -> narrowed down from 24 candidate to only 6. • Binding affinity determination via BIAcore 3000 (SPR): CSIR 2.19 has the lowest KD (1.6±0.5nM). • Competitive aptamer-antibody ELONA assay – CSIR 2.11 and CSIR 2.19 are unaffected (binding site is different). CSIR 2.11 has a KD of 8±1.07nM; CSIR 2.19 has a KD of 1.6±0.5nM Whattheyhavedone?
- 10. • In order to determine which part of the heterodimer did the aptamers bind to… they did another ELONA. • They noted that despite being selected against the whole heterodimer, the aptamers have shown to recognise CFP-10 but NOT ESAT-6. Whattheyhavedone?
- 11. What have they done? • Specificity testing All aptamers are able to recognise MTB lysates None are binding to S. pyrogenes or C. xerosis 5/6 aptamers recognise M. smegmatis, M. bovis (BCG) and S. aureus lysates. 3/6 aptamers recognise P. aeruginosa lysate
- 12. What have they done? Antigen detection in sputum sample • CSIR 2.11 tested in 68 sputum samples via ELONA • Cut-point: At OD450 = 0.2 (for Youden’s Index)
- 13. Differentiating specificity and sensitivity
- 14. Statistics
- 15. • Youden's J statistic (a.k.a. Youden's index) is a single statistic that captures the performance of a diagnostic test. • May not correspond to cut-points that rules in or rules out disease. • BUT is a chance corrected measure and is better interpretable across different problems and populations than easily biased measures like recall and precision, accuracy or F-score. • Rule-in disease cut-point • I’m assuming that this is the more accurate cut-point since it rules in the disease unlike Youden’s which does not discriminate whether the subject actually has the disease or not Why did they use 2 indexes? • The use of a single index is "not generally to be recommended” (Everitt, 2002) Statistics J = Sensitivity + Specificity − 1
- 16. Discussions and limitations • Even though aptamers are raised against the heterodimer, the aptamers have shown a preference for CFP-10. But they explained that this is perhaps because ESAT-6 binding site is not accessible due to it being bound to CFP-10. And when compared with their previous experiment using RNA aptamers, they conclude that “perhaps RNA aptamers form more stable secondary and tertiary structures than ssDNA aptamers” This leads to the results as seen in their ‘Specificity Testing’. Their aptamer is able to bind to the CFP-10 monomer that is secreted by a number of cells. For cells that do not produce CFP-10 (S. aureus), they do produce other ESAT-6 homologues (like Esx A and Esx B proteins). • Cross-reactivity of the aptamers can be improved via optimisation of the aptamer (truncation)
- 17. Questions unanswered • They did not give a reason as to why they choose aptamer CSIR 2.11. • CSIR 2.19 seems to be a better candidate – lowest KD among the other 6 aptamers, have the same results as CSIR 2.11 for specificity testing. • Maybe because CSIR 2.11 binds a tiny bit to ESAT-6?
- 18. Further work • Since the flexible C-terminus tail of CFP-10 is the main component of the complex in the binding to WBC, they can check and see if their aptamer somehow inhibited the function of the CFP-10 tail. • More optimisation is needed to improve performance outcomes, such as the optimisation of the aptamer • Needs better validation by using a larger clinical sample size and from different parts of the body
- 19. Conclusion • Based on the specificity (68.75%) and sensitivity (100%) obtained using the Youden’s Index, they concluded that it is encouraging for the development of aptamers as detection reagent for TB • Compared with their previous experiment using RNA aptamers, they conclude that “perhaps RNA aptamers form more stable secondary and tertiary structures than ssDNA aptamers” • Binding affinity of their selected aptamer CSIR 2.11 has binding affinity in the nanomolar range. • ELONA lacks rapid readout – it takes couple of hours to perform ELONA. • Labeled this as a preliminary proof of concept study on the feasibility of using aptamers for sputum samples
- 20. Further reading • Youden Index and optimal cut-point estimated from observations affected by a lower limit of detection - http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2515362/ • Optimal cut-point and its corresponding Youden Index to discriminate individuals using pooled blood samples - http://www4.stat.ncsu.edu/~bondell/OptimCut.pdf • Revisiting Youden’s Index as a useful measure of the misclassification error in meta-analysis of diagnostic studies - http://www.personal.soton.ac.uk/dab1f10/SMM081867.pdf
- 21. Thank you!