Abstract
This thesis provides an approach to identify and characterize the enormous diversity of Naegleria genus with Closed-Tube Barcoding. This thesis is comprised of two parts: 1) The Naegleria-Assay, which can be used to identify any species of Naegleria on earth and 2) The Fowleri-Assay, which can be used to selectively detect the “brain eating amoeba” Naegleria fowleri.\r \r In the construction of these assays, Closed-Tube Barcoding was used to generate fluorescent signatures of the CO1 gene for sequence variants of the genus Naegleria. The Naegleria-Assay can generate a unique fluorescent signature for each of the sixteen tested species of Naegleria and can even differentiate between different strains within a single species. With a few adjustments in thermal cycle and reagents, this assay can be transformed into the Fowleri-Assay, which can identify as few as ten copies of Naegleria fowleri in a background of up to 10,000 copies of DNA from other Naegleria species. The efficiency of this reaction is 91%. These assays indicate that a single set of probes can be used to characterize an entire diverse genus and can provide critical information about the diversity and range of the Naegleria genus as well as identify the pathogenic species Naegleria fowleri.