Molecular detection and identification of parasites involved in human disease
Master of Science
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The detection and identification of disease-causing parasites via DNA and the polymerase chain reaction (PCR) offers a new approach for quick and efficient disease diagnosis. Three nucleic acid based technologies have been developed and optimized for the identification of six species of parasites that cause disease, and designed to amplify small fragments of DNA likely to remain in archaeological material. Two methods involve a species-specific identification by examining four gene fragments, one of which has been designed as a multiplex PCR to minimize the time required for diagnosis. The third method involves the use of one species-specific gene target per parasite in a multiplex PCR for the detection of multiple parasites in one reaction to expedite the testing process and confirmation of the disease in question in a clinical setting. Sensitivity and specificity have been proven through the use of a 1/1000 diluted DNA template and sequencing of products confirmed the presence o f each species analyzed. With increased sensitivity, a less invasive sampling procedure would be required from the patient. Also, the earlier the presence of parasites in the tissues is detected, the earlier the diagnosis would occur. Tissues that contain very few parasites and had been classified as previously unusable for diagnosis or tissues from older medical cases can be analyzed for the presence of disease and the identification of causative species. The successful application of the singleplex PCR technique to Plasmodium falciparum and Leishmania degraded DNA samples leads to the feasibility of success for the multiplex PCR techniques involving degraded DNA analyses. Therefore, this methodology may be applied to samples of unknown disease content to either include or exclude the agents of disease. Future directions may extend these analyses to include a range of other agents of disease, thus allowing for the determination of disease antiquity through degraded DNA analyses.