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

Abstract

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.

Species identification of marine invertebrate early stages by whole-larvae in situ hybridisation of 18S ribosomal RNA

“…Whole-cell in situ hybridisation methods using oligonucleotide probes targeting rRNA have successfully identified bacteria and archea (DeLong et al 1989, Amann et al 1990, Pernthaler et al 2002, diatoms (Scholin et al 1997), nanoflagellates (Lim et al 1996), Microsporidia (Hester et al 2000), ciliates (Petroni et al 2002) and picophytoplankton (Simon et al 2000). Despite the clear advantage of this technique for identifying larval stages, surprisingly, there has been only 1 attempt to extend it to marine larvae (Goffredi et al 2006).…”

Simultaneous Detection of Four Human Pathogenic Microsporidian Species from Clinical Samples by Oligonucleotide Microarray

“…Although immunofluorescent staining techniques have been developed for species differentiation, they are far from optimal due to problems of limited availability of antibodies, nonspecific binding, and interference from the sample matrix (1,23). The fluorescence in situ hybridization technique has been reported to detect microsporidian species in environmental and clinical samples (3,17,30) and has shown great sensitivity and specificity, but its performance requires expertise and a long hybridization time, and it has only been occasionally used in practice.…”

Retrospective Species Identification of Microsporidian Spores in Diarrheic Fecal Samples from Human Immunodeficiency Virus/AIDS Patients by Multiplexed Fluorescence In Situ Hybridization

“…The fluorescence in situ hybridization (FISH) assay uses fluorescently labeled 19-bp oligonucleotide probes targeted to microsporidium species-specific sequences of 16S rRNA; therefore, spore identification is species specific (18,19,21,31). The FISH assay was originally developed for E. hellem (21); however, alignment of the respective 16S rRNA regions of 22 other species of microsporidia (21) allowed the design of oligonucleotide probes specific to E. intestinalis (18) and to E. bieneusi and E. cuniculi (19).…”

“…The fluorescence in situ hybridization (FISH) assay uses fluorescently labeled 19-bp oligonucleotide probes targeted to microsporidium species-specific sequences of 16S rRNA; therefore, spore identification is species specific (18,19,21,31). The FISH assay was originally developed for E. hellem (21); however, alignment of the respective 16S rRNA regions of 22 other species of microsporidia (21) allowed the design of oligonucleotide probes specific to E. intestinalis (18) and to E. bieneusi and E. cuniculi (19). Through the use of various fluorochromes for probe labeling, E. bieneusi, E. intestinalis, E. hellem, and E. cuniculi spores are stained in yellow, red, green or blue, and orange, respectively (18,19,21,31), which facilitates the simultaneous use of all four probes, i.e., multiplexing.…”

“…The FISH assay was originally developed for E. hellem (21); however, alignment of the respective 16S rRNA regions of 22 other species of microsporidia (21) allowed the design of oligonucleotide probes specific to E. intestinalis (18) and to E. bieneusi and E. cuniculi (19). Through the use of various fluorochromes for probe labeling, E. bieneusi, E. intestinalis, E. hellem, and E. cuniculi spores are stained in yellow, red, green or blue, and orange, respectively (18,19,21,31), which facilitates the simultaneous use of all four probes, i.e., multiplexing. The multiplexed approach has been successfully applied to testing freshly collected environmental samples (19) and animal, i.e., bird, fecal samples (31).…”