The use of a ribosomal RNA targeted oligonucleotide probe for fluorescent labelling of viable Cryptosporidium parvum oocysts.
A fluorescence in situ hybridization (FISH) technique has been developed for the fluorescent labelling of Cryptosporidium parvum oocysts in water samples. The FISH technique employs a fluorescently labelled oligonucleotide probe (Cry1 probe) targeting a specific sequence in the 18S ribosomal RNA (rRNA) of C. parvum. Hybridization with the Cry1 probe resulted in fluorescence of sporozoites within oocysts that were capable of excystation, while oocysts that were dead prior to fixation did not fluoresce. Correlation of the FISH method with viability as measured by in vitro excystation was statistically highly significant, with a calculated correlation coefficient of 0.998. Examination of sequence data for Cryptosporidium spp. other than C. parvum suggests that the Cry1 probe is C. parvum-specific. In addition, 19 isolates of C. parvum were tested, and all fluoresced after hybridization with the Cry1 probe. Conversely, isolates of C. baileyi and C. muris were tested and found not to fluoresce after hybridization with the Cry1 probe. The fluorescence of FISH-stained oocysts was not bright enough to enable detection of oocysts in environmental water concentrates containing autofluorescent algae and mineral particles. However, in combination with immunofluorescence staining, FISH enabled species-specific detection and viability determination of C. parvum oocysts in water samples.
Vesey
G
G
Macquarie University Centre for Analytical Biotechnology, School of Biological Sciences, Macquarie University, New South Wales, Australia. gvesey@rna.bio.mq.edu.au
Ashbolt
N
N
Fricker
E J
EJ
Deere
D
D
Williams
K L
KL
Veal
D A
DA
Dorsch
M
M
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002463:Cell Membrane Permeability; D016785:Cryptosporidium parvum / Q000235:genetics / Q000302:isolation & purification*; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D005784:Gene Amplification; D017404:In Situ Hybridization, Fluorescence / Q000379:methods* / Q000592:standards; D015345:Oligonucleotide Probes; D012329:RNA, Bacterial / Q000032:analysis; D012337:RNA, Ribosomal, 18S / Q000032:analysis; D012680:Sensitivity and Specificity; D013194:Staining and Labeling; D014881:Water Supply
D000911:Antibodies, Monoclonal; D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D012329:RNA, Bacterial; D012337:RNA, Ribosomal, 18S; D016650:Fluorescein-5-isothiocyanate