Rapid method for fluorescent in situ ribosomal RNA labelling of Cryptosporidium parvum.
A method for fluorescence in situ hybridization (FISH) is described that requires less than 1 h duration. Oocysts were resuspended in 50% ethanol and incubated at 80 degrees C for 10 min for simultaneous fixation and permeabilization. Samples were than incubated with the oligonucleotide probe at 48 degrees C for more than 30 min. The rRNA binding specificity of the optimized protocol was confirmed. FISH was found to be valuable as a second label for oocysts presumptively identified immunofluorescently, but required more than an order of magnitude signal amplification for independent use. The number of oligonucleotide probes bound per oocyst was compared with the copy number of 18S rRNA molecules per oocyst to provide a measure of the labelling efficiency of the FISH method. Hybridization kinetics were also analysed. These data indicate that significant further increases in the brightness of FISH-labelled oocysts cannot be achieved by further optimization of the pre-treatment and hybridization conditions.
Deere
D
D
Macquarie University Centre for Analytical Biotechnology, School of Biological Sciences, Sydney, Australia.
Vesey
G
G
Milner
M
M
Williams
K
K
Ashbolt
N
N
Veal
D
D
D000818:Animals; D000911:Antibodies, Monoclonal; D016785:Cryptosporidium parvum / Q000235:genetics / Q000302:isolation & purification*; D005434:Flow Cytometry; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D016014:Linear Models; D016053:RNA, Protozoan / Q000032:analysis*; D012335:RNA, Ribosomal / Q000032:analysis*; D012337:RNA, Ribosomal, 18S / Q000032:analysis; D013997:Time Factors
D000911:Antibodies, Monoclonal; D016053:RNA, Protozoan; D012335:RNA, Ribosomal; D012337:RNA, Ribosomal, 18S