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FLUORESCENT IN SITU DETECTION OF ENCEPHALITOZOON HELLEM SPORES WITH A 6-CARBOXYFLUORESCEIN-LABELED RNA-TARGETED OLIGONUCLEOTIDE PROBE
Citation:
Hester, J. D., H.D A. Lindquist, A. M. Bobst, AND F W. Schaefer. FLUORESCENT IN SITU DETECTION OF ENCEPHALITOZOON HELLEM SPORES WITH A 6-CARBOXYFLUORESCEIN-LABELED RNA-TARGETED OLIGONUCLEOTIDE PROBE. May-June, JOURNAL OF EUKARYOTIC MICROBIOLOGY 47(3):299-308, (2000).
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:
A fluorescent in situ hybridization assay has been developed for the detection of the human-pathogenic microsporidian, Encephalitozoon hellem, in water samples using epifluorescence microscopy. The assay employs a 19-nucleotide species-specific 6-carboxyfluorescein-labeled oligonucleotide probe, HEL878F, designed to be complementary to the nucleic acid sequence 878-896, a highly variable segment of the 16S ribosomal RNA of E. hellem spores. The specificity of this probe for its ribosomal RNA target site was confirmed using RNA degradation, ribosomal RNA target site competition, and nucleotide base mismatch control probe assays. Furthermore, the specificity of the HEL878F oligonucleotide probe for E. hellem spores was established when it was evaluated on spores from all three species of the genus Encephalitozoon that had been seeded in reagent water and environmental water concentrates. The specificity of the HEL878F oligonucleotide probe was further corroborated when tested on algae, bacteria, and protozoa commonly found in environmental water. The study demonstrates the applicability of a fluorescent in situ hydridization assay using a species-specific fluorescent-labeled oligonucleotide probe for the detection of E. hellem spores in water samples.