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Species-Specific Detection of Three Human-Pathogenic Microsporidial Species from the Genus Encephalitozoon Via Fluorogenic 5' Nuclease Pcr Assays

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Abstract:This describes fluorogenic 5' nuclease PCR assays suitable for rapid, sensitive, quantitative, high-throughput detection of the human-pathogenic microsporidial species Encephalitozoon hellem, E. cunicli and E. intestinalis. The assays utilize species-specific primer sets and a genus-specific dual fluorescent-labeled probe that anneals to a region within the Encephalitozoon 16S rRNA gene. The assay design permits the probe to be used either with one set of primers for species-level determination or with a combination of all three primer sets for a genus-level screening of samples.

The linear range of all three species-specific calibration curves that were developed using serial ten-fold diultions of genomic DNA isolated form hemacytometer counted spores was determined to be between 10 +4 minus 10 -1 spores per PCR sample. The coefficients of variation were less than or equal to 5% over the entire 5-log span of each calibration curve. When DNA isolated from flow cytometric enumerated spores from each of the three Encephalitozoon species was used to evaluate the quantitative capability of the species' respective calibration curves, the results from 34 out of 36 (94%) samples were within 2 standard deviations. The species-specificity of each assay was confirmed using DNA isolated from 10 +4 spores from each of the other two Encephalitozoon species as well as DNA extracted from numerous other protozoa, algae and bacteria.
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Citation:Hester, J. D., M. Varma, A. M. Bobst, M. W. Ware, H. A. Lindquist, and F. W. Schaefer. Species-Specific Detection of Three Human-Pathogenic Microsporidial Species from the Genus Encephalitozoon Via Fluorogenic 5' Nuclease Pcr Assays. MOLECULAR AND CELLULAR PROBES 16(6):435-444, (2002).
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Contact: Mary P. O'Bryant - (919)-541-4871 or obriant.mary@epa.gov
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Division: Microbiological & Chemical Exposure Assessment Division
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Branch: Biohazard Assessment Research Branch
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Product Type: Journal
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Published: 12/01/2002
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Related Entries:
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Bullet Item Detecting Ccl-Related, Emerging and Regulated Waterborne Human Protozoa for Exposure Assessment
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Last Updated on Monday, October 22, 2007
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