Paper
Three-color Cytometry for the Simultaneous Detection of the Cryptosporidium Species Contributing to the Majority of Human Cryptosporidiosis
- Authors:
- Anitha Alagappan; Peter Bergquist; Belinda Ferrari
- Abstract
- Cryptosporidium is a protozoan parasite which is the most common non-viral cause of diarrhea worldwide. Currently, there are twenty recognized species of Cryptosporidium and among which, C. parvum and C. hominis are the species primarily infecting humans. FISH utilises fluorescently-labelled complementary DNA oligonucleotide probes that target specific sequences of cellular rRNA for direct identification of microorganisms. This work describes the successful development of such probes for the specific detection of the zoonotic species, C. parvum and the human host-specific species, C. hominis. The specificity of the probes was established by testing them against a range of target and non-target Cryptosporidium species using an optimised FISH assay. Validation of the C. parvum–specific probe and C. hominis-specific probe was carried out by a comparison of FISH with PCR-RFLP analysis of the 18S rRNA gene from Cryptosporidium isolated from human infections. Additionally, the C. parvum-specific probe synthesised with Cy3 dye and the C. hominis-specific probe synthesised with Cy5 dye were used in combination with the Cryptosporidium-specific monoclonal antibody CRY104-FITC, for the development of a 3-colour FISH assay that can allow identification of both C. parvum and C. hominis species simultaneously. This assay was developed and validated by using both epi-fluorescence microscopy and flow cytometric technology. Species-level identification by three-color cytometry and PCR-RFLP targeting the 18S rRNA gene was identical. The potential exists for the assay to be implemented into routine immunofluorescence detection methods for Cryptosporidium.
- Keywords
- Cryptosporidium; Fluorescence in Situ Hybridization; Monoclonal Antibodies; Flow Cytometry; Three-color
- StartPage
- 103
- EndPage
- 108
- Doi
- 10.5963/PHF0202007