Rapid method for fluorescentin situribosomal RNA labelling ofCryptosporidium parvum

Abstract

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°C for 10 min for simultaneous fixation and permeabilization. Samples were then incubated with the oligonucleotide probe at 48°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.

Comparative study between two laser scanning cytometers and epifluorescence microscopy for the detection of Cryptosporidium oocysts in water

“…A variety of techniques is being used to detect oocysts in such water concentrates, including PCR (8)(9)(10), RT-PCR (11), fluorescent ''in situ hybridization'' (12), enzyme linked immunosorbant assay (13,14), and cell culture infectivity (15). However, most studies and water laboratories continue to rely on immunofluorescence techniques, which involve the incubation of the sample with a fluoresceinisothiocyanate conjugated Cryptosporidium spp.…”

Evaluation of fluorochromes for flow cytometric detection of Cryptosporidium parvum oocysts labelled by fluorescent in situ hybridization

“…Two were complementary to 18S rRNA regions : 'CRY1' (21-mer, 5?-CGG TTA TCC ATG TAA GTA AAG-3?, apparently specific for C. parvum, Vesey et al 1998) and 'EUK' (16-mer, 5?-ACC AGA CTT GCC CTC C-3?, conserved for all Eukarya, Amann et al 1990). A negative control, 'ANTI-EUK' (16-mer, 5?-G GAG GGC AAG TCT GGT-3?, Deere et al 1998b) was the complement of EUK. Probes were synthesized with a 5?-amino linker and subsequently conjugated to a range of fluorochromes that were efficiently excited at 488 nm.…”

“…Probe concentrations were determined using u.v. spectrophotometry (Sambrook et al 1989) and labelling efficiency checked using spectrophotometry as described by Deere et al (1998b). Oligonucleotide probes were allowed to hybridize with target rRNA as described by Deere et al (1998b).…”

“…RNAase treated controls were prepared as described by Deere et al (1998a). Cells, spores and oocysts were fixed and permeabilized using ethanol/heat treatment (Deere et al 1998b). …”

“…Recently, Deere et al (1998b) found that optimization of oocyst pre-treatment was not able to increase signal intensity beyond the levels reached by Vesey et al (1998) as the number of target ribosomes appeared to be the limiting factor. Therefore, the signal per ribosome must be increased to boost signal intensities.…”