| Description |
Molecular detection of Tropheryma whipplei is an important tool in the diagnosis of Whipple's Disease and other T. whipplei infections. This thesis describes the design and validation of a real-time polymerase chain reaction assay that detects T. whipplei in cerebrospinal fluid, whole blood, tissue, formalin-fixed paraffin-embedded tissue, serum, and plasma samples. The assay detects a repeated sequence present seven times in the T. whipplei genome and the rpoB gene in a multiplex format. The assay is highly specific for T. whipplei and is capable of detecting less than four copies per PCR reaction with certain sample types. The validated assay has been in use at ARUP Laboratories since April 2016 and has detected T. whipplei in three patient samples. This thesis also describes a method for manufacturing formalin-fixed paraffin- embedded blocks containing cultured bacteria as a means of simulating positive patient samples when genuine positive samples are not available. However, quantification of the number of bacteria in a sample using this method requires postextraction quantification of target copy number. Digital polymerase chain reaction is capable of absolute quantification of a target sequence without reference to a standard curve and was used to quantify target copy number in manufactured formalin-fixed paraffin-embedded samples. The World Health Organization produces International Standards which serve as a common calibration material for many types of diagnostic testing. This includes several quantification standards for various pathogens which are quantified by molecular methods. However, these standards are not well characterized in terms of true copy number and nucleic acid sequence, which can lead to discordant results between molecular assays targeting different regions of a given pathogen's genome. This thesis also describes the use of digital polymerase chain reaction to evaluate the World Health Organization 1st International Standard for Epstein-Barr virus and the World Health Organization 1st International Standard for Hepatitis D virus. This provides better characterization of each standard by estimating the viral copy number. |
