You are here:
SPECIES-SPECIFIC DETECTION OF THREE HUMAN-PATHOGENIC MICROSPORIDIAL SPECIES FROM THE GENUS ENCEPHALITOZOON VIA FLUOROGENIC 5' NUCLEASE PCR ASSAYS
Citation:
Hester, J. D., M Varma, A. M. Bobst, M W. Ware, H.D 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).
Impact/Purpose:
1) Refine new, practical methods for the detection of CCL-related and emerging waterborne human protozoa.
2) Perform field tests of devices or methods that have been developed under this task.
3) Evaluate these methods or devices in a variety of water matrices and parasite concentrations.
This work in this task supports CCL2 and 3 and is expected to be completed by 9/07.
Description:
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.