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Tuberculosis (Edinb). 2018 Sep;112:27-36. doi: 10.1016/j.tube.2018.07.004. Epub 2018 Jul 10.

Generation and application of DNA aptamers against HspX for accurate diagnosis of tuberculous meningitis.

Author information

1
Department of Biotechnology, All India Institute of Medical Sciences (AIIMS), New Delhi 110029, India; Faculty of Pharmacy, Uttarakhand Technical University (UTU), Dehradun 248007, Uttarakhand, India.
2
Centre for Biodesign and Diagnostics, Translational Health Science and Technology Institute (THSTI), Faridabad 121001, Haryana, India.
3
Discipline of Biosciences and Biomedical Engineering, Indian Institute of Technology Indore, Simrol, Indore 453552, India.
4
Department of Biochemistry, Dr. Ram Manohar Lohia Hospital, New Delhi 110001, India.
5
Department of Pediatrics, Dr. Ram Manohar Lohia Hospital, New Delhi 110001, India.
6
Faculty of Pharmacy, Uttarakhand Technical University (UTU), Dehradun 248007, Uttarakhand, India.
7
Centre for Biodesign and Diagnostics, Translational Health Science and Technology Institute (THSTI), Faridabad 121001, Haryana, India; AptaBharat Innovation Private Limited, Translational Health Science and Technology Institute Incubator, Faridabad 121001, Haryana, India. Electronic address: tarun@thsti.res.in.
8
Department of Biotechnology, All India Institute of Medical Sciences (AIIMS), New Delhi 110029, India; Centre for Biodesign and Diagnostics, Translational Health Science and Technology Institute (THSTI), Faridabad 121001, Haryana, India. Electronic address: jayatyagi.aiims@gmail.com.

Abstract

Tuberculous meningitis (TBM) is the most severe manifestation of tuberculosis and its diagnosis remains a challenge even today due to the lack of an adequate test. HspX antigen of Mycobacterium tuberculosis was previously established as a reliable diagnostic biomarker for TBM in an ELISA test format using anti-HspX polyclonal antibodies. Towards overcoming the limitations of batch-to-batch variation and challenges of scalability in antibody generation, we utilized Systematic Evolution of Ligands by EXponential enrichment (SELEX) to develop high affinity DNA aptamers against HspX as an alternative diagnostic reagent. Post-SELEX optimization of the best-performing aptamer candidate, H63, established its derivative H63 SL-2 M6 to be superior to its parent. Aptamer H63 SL-2 M6 displayed a specific and high affinity interaction with HspX (Kd ∼9.0 × 10-8 M). In an Aptamer Linked Immobilized Sorbent Assay (ALISA), H63 SL-2 M6 significantly differentiated between cerebrospinal fluid specimens from TBM and non-TBM subjects (n = 87, ***p < 0.0001) with ∼100% sensitivity and ∼91% specificity. Notably, ALISA exhibited comparable performance with previously reported antibody-based ELISA and qPCR. Altogether, our findings establish the utility of HspX aptamer for the reliable diagnosis of TBM and pave the way for developing an aptamer-based point-of-care test for TBM.

KEYWORDS:

Aptamer; Aptamer linked immobilized sorbent assay; Detection; Mycobacterium tuberculosis; Tuberculous meningitis

PMID:
30205966
DOI:
10.1016/j.tube.2018.07.004

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