. The purpose of this study is to optimize the DNA-based methods for species-specific diagnoses of microsporidiosis. The first specific aim will be to define the optimal methods for sample preparation, extraction of microsporidian DNA, and amplification of DNA by PCR for improving consistency in obtaining microsporidian DNA from clinical specimens (primarily stool and urine.) The second specific aim will be to define the levels of sensitivity, specificity, and reliability of methods used for detection of PCR DNA products. The detection methods that will be used include, ethidium bromide staining of PCR products, restriction fragment length polymorphism (PCR-RFLP), double-stranded DNA heteroduplex mobility shift analysis, and Southern analysis. The P.I. believes that the DNA-based methods incorporating the polymerase chain reaction (PCR) should result in more sensitive, specific and reliable levels of diagnosis than the histochemistry methods now being used, and will be important in epidemiology for defining the source and spread of microsporidia infections as ways to reduce exposure or prevent infection. She adds that species identification will be important for therapeutics because different species of microsporidia express different levels of sensitivity to some drugs (eg. albendazole) now being used to treat microsporidiosis.
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