Fluorescent in situ Detection of Encephalitozoon hellem Spores with a 6-Carboxyfluorescein-Labeled Ribosomal RNA-Targeted Oligonucleotide Probe

• Published in: The Journal of eukaryotic microbiology Vol. 47; no. 3; pp. 299 - 308
• Main Authors: Hester, Jeff D, Alan Lindquist, H. D, Bobst, Albert M, Schaefer, Frank W
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01-05-2000; Blackwell
• Subjects: AIDS; Animals; Biological and medical sciences; Encephalitozoon - classification; Encephalitozoon - genetics; Encephalitozoon - isolation & purification; environmental water concentrate; epifluorescence microscopy; FISH; Fluoresceins; fluorescent in situ hybridization; Fundamental and applied biological sciences. Psychology; General aspects and techniques; human-pathogenic; In Situ Hybridization, Fluorescence; microsporidia; Oligonucleotide Probes; Protozoa; RNA, Ribosomal, 16S - isolation & purification; rRNA; Sensitivity and Specificity; species-specific; Water - parasitology; waterborne; Ohio; United States; North America; America; Water; Human; Protozoa; Host parasite relation; Protozoal disease; Methodology; Parasite; Species specificity; Parasitosis; Method; Spores; Cnidosporidia; Infection; Freshwater environment; Ribosomal RNA; Fluorescein; Fluorescence in situ hybridization; Microsporidiosis; Stream; Oligonucleotide; Molecular probe; Detection
• ISSN: 1066-5234; 1550-7408
• DOI: 10.1111/j.1550-7408.2000.tb00051.x

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 hybridization assay using a species-specific fluorescent-labeled oligonucleotide probe for the detection of E. hellem spores in water samples.