A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Food Engineering and Bioprocess Technology, School of Environment, Resources and Development

Thesis (M. Sc.) - Asian Institute of Technology, 2017

A sensitive, simple and inexpensive technique for point - of - care (POC) diagnosis of hepatitis B (HBV) is highly desirable majorly due to the lack of advanced medical and diagnostic facilities in some endemic areas . The objective of this study was to develop a low - cost and simple device based on Loop - mediated isothermal amplification (LAMP) of DNA and electrochemical measurement for rapid detection of HBV. The electrochemical mediato r methylene blue (MB) was intercalated into the LAMP amplicons and square wave voltammetry (SWV) signals were processed by a hand - held potentiostat. SWV response of MB was studied with different concentrations. LAMP was optimized for temperature and MB con centrations for real time monitoring. Quantification curves were developed followed by testing of unknowns, using conventional and sigmoidal model fitting (4 parametric) approaches whereas both peak height ratio (PHR) and amplification times were used as t hresholds. Sensitivity and specificity were assessed followed by testing human plasma without DNA extraction. A positive linear relationship of peak currents of SWV vs concentrations of MB was observed with significantly higher currents at higher temperat ure. Incubation temperature of 63 o C and 25 o M of MB were the optimized condition for LAMP based on reduction of PHRs. Linear relationships between logarithmic concentrations of HBV DNA vs amplification time and PHRs depending on the thresholds were obtain ed with different coefficient of determination (R 2 ) value. Peak height thresholds of 0.6 and time threshold of 22 min were found to be more suited in conventional approach while the former varied to 0.7 and latter remined same in model fitting approach, ba sed on R 2 . Concentrations of the unknowns were quantified successfully to the logarithmic concentrations of DNA. The a pproach was found to be specific for HBV as no PHR reduction for human genomic DNA and HBV DNA up to fg/ o l was detected . Human plasma coul d be used directly as the temp late after a pre - heat treatment of either subsequent heating for 5 min at 95 o C and 100 o C or 125 o C for 10 min whereas the centrifugation step of the former increased the LAMP efficiency.