Evaluation Of Advanced Disease Diagnostic Methods by Using Nucleic Acids

Pathogenic organisms are transmitted to the host organism through all possible connected
pathways, and cause severe disease state in the new host. Commonly occurring curable infectious diseases still
impose the greatest health impacts on a worldwide perspective. Several technologies are used for diagnosing the
disease state in that particular host. Golden standard for diagnostics of pathogenic bacteria has long been culture
able Medias. This method was used from the ancient days to diagnose a disease. Environmental biologists have
estimated that less than 1% of all bacteria are culture able and it takes lots of time to get conformation about the
disease, severity and causative organism. And these are the cost effective techniques. Genomic-based approaches
offer the potential to identify all microbes from all the biological kingdoms. Nucleic acid based pathogen
diagnostics has evolved significantly over the past decades. Novel technologies offer increased potential in
sensitivity, specificity, decreased costs and parallel sample management. However, most methods are confined to
core laboratory facilities. To construct an ultimate nucleic acid based diagnostic for use in areas of need, potential
frontline techniques need to be identified and combined.
The research focus of this work has been to develop and apply nucleic acid based methods for pathogen
diagnostics. Methods and assays were applied to the two distinct systems i) screening for antibiotic resistance
mutations in the bacterial pathogen Helicobacter pylori appears to be an important risk factor for stomach ulcers,
gastritis and possibly stomach cancer and ii) genotype determination of the cancer causative Hepatitis B and C
virus (HBV & HCV). The first part of the study included development of rapid, direct and multiplex Pyro
sequencing nucleic acid screenings. With improved methodology in the sample preparation process, we could
detect an existence of multiple co-infecting HBV & HCV genotypes at greater sensitivities than previously
described, when using the same type of methodology. The second part of the study focused on multiplex nucleic
acid amplification strategies using Molecular Inversion Probes with end-step Pyro sequencing screening.