Low copy number quantification of DNA utilising Loop-mediated Amplification (LAMP) with Bioluminescent Assay in Real-Time (BART) reporter

iv Abstract Low Copy Number Quantification of DNA Utilising Loop - mediated Amplification (LAMP) with Biolumines cent Assay in Real - Time (BART) Reporter Real time quantitative PCR is the benchmark technology of molecular diagnostics in a wide range of fields including forensic science, clinical diagnosis and the detection of genetically modified (GM) c rops. T here is a requirement for rapid, cheap and simple portable quantitative and specific diagnostics . Quantitative PCR is limited by a number of factors in this regard: t he complex hardware is often expensive and largely laboratory limited. Bioluminescent Assay in Re al Time (BART) is a nucleic acid amplification detection system that converts inorganic pyrophosphate (PPi) , a by - product of DNA synthesis , into light output. The pyrophosphate is converted into ATP which is utilised by a thermostable luciferase to convert luciferin to oxyluciferin with the emission of light. The development of isothermal amplification techniques that use the strand displacement properties of certain DNA polymerases enables the BART detection to be utilised in simple and cheap hardware at a single temperature. Loop - mediated amplification (LAMP) is an isothermal amplification method which is highly specific to the DNA target sequence and produ ces high concentrations of PPi