PMID- 9830116
OWN - NLM
STAT- MEDLINE
DCOM- 19981218
LR  - 20190915
IS  - 1364-5072 (Print)
IS  - 1364-5072 (Linking)
VI  - 85
IP  - 5
DP  - 1998 Nov
TI  - Rapid method for fluorescent in situ ribosomal RNA labelling of Cryptosporidium 
      parvum.
PG  - 807-18
AB  - 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.
FAU - Deere, D
AU  - Deere D
AD  - Macquarie University Centre for Analytical Biotechnology, School of Biological 
      Sciences, Sydney, Australia.
FAU - Vesey, G
AU  - Vesey G
FAU - Milner, M
AU  - Milner M
FAU - Williams, K
AU  - Williams K
FAU - Ashbolt, N
AU  - Ashbolt N
FAU - Veal, D
AU  - Veal D
LA  - eng
PT  - Comparative Study
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - England
TA  - J Appl Microbiol
JT  - Journal of applied microbiology
JID - 9706280
RN  - 0 (Antibodies, Monoclonal)
RN  - 0 (RNA, Protozoan)
RN  - 0 (RNA, Ribosomal)
RN  - 0 (RNA, Ribosomal, 18S)
SB  - IM
MH  - Animals
MH  - Antibodies, Monoclonal
MH  - Cryptosporidium parvum/genetics/*isolation & purification
MH  - Flow Cytometry
MH  - In Situ Hybridization, Fluorescence/*methods
MH  - Linear Models
MH  - RNA, Protozoan/*analysis
MH  - RNA, Ribosomal/*analysis
MH  - RNA, Ribosomal, 18S/analysis
MH  - Time Factors
EDAT- 1998/11/27 00:00
MHDA- 1998/11/27 00:01
CRDT- 1998/11/27 00:00
PHST- 1998/11/27 00:00 [pubmed]
PHST- 1998/11/27 00:01 [medline]
PHST- 1998/11/27 00:00 [entrez]
AID - 10.1046/j.1365-2672.1998.00589.x [doi]
PST - ppublish
SO  - J Appl Microbiol. 1998 Nov;85(5):807-18. doi: 10.1046/j.1365-2672.1998.00589.x.