Development of Biosensors Using Aptamers and Nucleic Acid Characteristics for Diagnosis of Tuberculosis

Abstract

According to the World Health Oganization (WHO), tuberculosis is still one of the most common infectious diseases worldwide. Although many diagnostic methods have been studied to prevent occurrence and transmission for tuberculosis, diagnostic methods that can be used without difficulty in underdeveloped countries tuberculosis patients are concentrated have not yet been developed. Based on the End tb strategy promulgated by the WHO, various studies such as diagnosis, treatment, and prevention methods for eradicating tuberculosis are ongoing. Most diagnostic methods currently used require Mycobacterium tuberculosis. Since there is a risk of secondary infection during the diagnosis process, the development of a diagnostic method excluding Mycobacterium tuberculosis is becoming important. In this Doctoral Thesis, for this purpose, aptamer was selected as a diagnostic probe and ESAT6 as a biomarker to overcome these limitations. Before showing the development process and results, Chapter Ⅰ introduces aptamer, its screening method, and additional nucleic acid- based diagnostic strategies. Chapters Ⅱ and Ⅲ show the development process and research of diagnostic methods using this. A diagnostic method that combines aptamer as a probe and nucleic acid characteristics enables improved diagnosis of current diagnostic methods. Chapter Ⅱ. Safe and sensitive detection of ESAT6 in non-infectious clinical samples via an aptamer-based qPCR strategy for tuberculosis diagnosis Tuberculosis (TB) is one of the leading causes of infection-related deaths worldwide. Conventional diagnostic methods for TB require infectious samples and are time - consuming. In Chapter Ⅱ, I aimed to develop an aptamer-based qPCR (Apta-qPCR) assay to diagnose TB safely and rapidly. The assay uses a single-stranded DNA aptamer with affinity for a TB biomarker, ESAT6, and molecular beacon capable of hybridizing to the aptamer. Apta-qPCR enables aptamer-mediated detection of ESAT6 via qPCR, wherein the aptamer serves as both a target recognition agent and template for quantification. This diagnostic method achieved a desirable detection limit of 2.03 nM in buffer with high reproducibility and reliability. Importantly, this assay has an advantage over smear microscopy in that it reduces the risk of infecti on by avoiding the use of sputum-containing infectious pathogens. Chapter Ⅲ. Highly sensitive luminometric aptasensor based on toehold- mediated strand displacement to detect ESAT6 for diagnosis of tuberculosis In Chapter Ⅲ, I proposed a novel luminometric detection strategy by detection of ESAT6, a biomarker of TB, in serum. A toehold-mediated strand displacement (TMSD)-based aptamer probe was designed for the sensing system and optimized using sequence length variation. Direct detection of ESAT6 was possible using an aptamer probe, and TMSD was applied as a one-step reaction. Following the optimization, the established aptasensor performed highly sensitive and specific detection of ESAT6 in buffer and serum, with detection limits of 0.66 and 0.62 pM, respectively. The diagnostic performance of the aptasensor was identified in a clinical application using serum samples from TB patients, with 78.38 % sensitivity, 93.33 % specificity, and 84.32 % accuracy based on receiver operator curve (ROC) analysis. The aptasensor diagnosed TB in a simple, economical method with high accuracy. In addition, ESAT6 was directly detected without additional steps by applying aptamer to the TMSD reaction. The developed luminometric aptasensor provided the possibility of a novel strategy for TB diagnosis using direct detection of ESAT6.