Hepatitis B Virus Genome Based Design Of A Genotyping And Drug Resistance Assay And An In House Viral Load Estimation Tool

Abstract

Hepatitis B virus infection is a global concern. India having around 4% of prevalence rate newlineis at growing risk of more number of deaths due to Hepatitis infection and other newlinecomplications associated with this disease. Availability of high end diagnostic tools that newlinecan detect HBV infection at an early stage, monitoring of patients diagnosed with infection newlineand management of treatment using advanced drug resistance tests are key issues which newlineare required to be addressed to combat the growing disease prevalence. newlineThis study aimed at development of such advanced diagnostic tools used clinical samples newlinefrom Indian patients. Use of home brew viral nucleic isolation method would reduce the newlinecost of testing, hence we aimed at development of a HBV DNA isolation method that is newlinesuperior to other laboratory developed methods and offers equivalent quality of DNA newlineisolation to that of commercially available kits. Through a well-designed and optimized newlinein-house HBV DNA isolation method, we were able to overcome the usual obstacle of newlinePCR inhibition and questionable reproducibility of laboratory developed DNA isolation newlinemethods. The unique combination of two lysis and two precipitation steps resulting in newlineenhance yield of DNA along with precipitation of potential PCR hindering molecules was newlinethe major availability through this method. As the in-house developed method matches newlineefficiency of commercially available DNA isolation kits and can be easily adapted for newlineroutine molecular diagnostics at comparatively lower price, this method holds lot of newlinepromises for implementation in resource limited diagnostic set ups. newlineEstimation of Hepatitis B viral load is required for detection of infection along with disease newlineprogression and therapy monitoring. We aimed to develop a viral load measurement newlinemethod that is equally significant to that of gold standard methods. The Hepatitis B viral newlineload assay method described in this thesis is robust, dependable, economical and effective newlinein resource limited settings like India. PCR probes specially designed from all H