Polymerase Chain Reaction-Single Strand Confirmation Polymorphism for the Detect

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There are many options for the detection and speciation of microsporidia in clinical specimens. Light microscopy allows detection of the parasites, but does not allow speciation. Electron microscopy is the gold standard for speciation, but is not as sensitive a method for the detection of microsporidia. Polymerase chain reaction (PCR) is a sensitive technique with many different methods for confirming a positive result and for determining the genus and species causing an infection. Speciation can be achieved by using species-specific primers in the PCR assay, or by using DNA probes or restriction endonucleases to determine the species after the PCR assay is performed. Single-strand confirmation polymorphism (SSCP) combined with PCR (PCR-SSCP) has been used to identify bacteria, fungi, and viruses. We will use our PCR assay for the detection of microsporidia in stool specimens and apply SSCP to determine the specific genus and species of the parasite. Organisms from cell culture will also be used to validate the method.The project has been expanded to apply SSCP to the detection of microsporidial genotypes in epidemiologic studies. Newly published primers allow speciation when the PCR products are digested with restriction enzymes. SSCP analysis will make this task easier and more sensitive than restriction enzyme analysis. We have identified two different genotypes among the Enterocytozoon bieneusi detected in patients with microsporidiosis at the NIH, which will be used to finish this study.This project has been completed.