Main content

Molecular diagnostic methods for Detection of Encephalitozoon cuniculi in pet rabbits

Show full item record

Title: Molecular diagnostic methods for Detection of Encephalitozoon cuniculi in pet rabbits
Author: Reabel, Stephanie
Department: Department of Pathobiology
Program: Pathobiology
Advisor: Weese, J.Scott
Abstract: Conventional methods such as serology and microscopy are unreliable for diagnosis of encephalitozoonosis in domestic rabbits. Previous studies have reported PCR to be insensitive but it is unclear whether this is because of inherent limitations or the lack of assay optimization and validation. The studies described in this thesis assess DNA quality and quantity for combinations of six DNA extractions kits and four spore disruption methods. The resulting DNA underwent PCR using a published primer set. The optimal method had a detection threshold of 100 spores/ml in saline. However, when repeated in urine, the detection threshold was much higher (10,000 spores/ml) and non-target DNA amplification was present. Various methods were used to improve analytical sensitivity and eliminate non-target amplification. One method involving PEG 8000 treatment produced a detection threshold of 1,000 spores/ml and decreased non-target DNA amplification. Ultimately, new primers were designed and when the optimized method was tested with these primers, a detection threshold of 100 spores/ml with no non-target DNA amplification was achieved. The optimal method and new primers were tested using clinical samples of rabbit urine and 32.4% were found to be positive for E. cuniculi. The final assay was shown to be both analytically sensitive and specific; however further clinical investigation is warranted to determine clinical utility.
URI: http://hdl.handle.net/10214/5275
Date: 2012-12


Files in this item

Files Size Format View
Reabel_Stephanie_201301_Msc.pdf 1.658Mb PDF View/Open

This item appears in the following Collection(s)

Show full item record