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

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Bibliographic Details
Published inJournal of applied microbiology Vol. 85; no. 5; pp. 807 - 818
Main Authors Deere, D., Vesey, G., Milner, M., Williams, K., Ashbolt, N., Veal, D.
Format Journal Article
LanguageEnglish
Published Oxford, UK Blackwell Science Ltd 01.11.1998
Blackwell Science
Oxford University Press
Subjects
Animals
Antibodies, Monoclonal
Biological and medical sciences
Cryptosporidium parvum
Cryptosporidium parvum - genetics
Cryptosporidium parvum - isolation & purification
Flow Cytometry
Fundamental and applied biological sciences. Psychology
General aspects and techniques
In Situ Hybridization, Fluorescence - methods
Linear Models
Protozoa
RNA, Protozoan - analysis
RNA, Ribosomal - analysis
RNA, Ribosomal, 18S - analysis
Time Factors
Protozoa
Apicomplexa
Rapid technique
Cryptosporidium parvum
Ribosomal RNA
Fluorescence in situ hybridization
Oocyst
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Summary: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.
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ISSN:1364-5072
1365-2672
DOI:10.1046/j.1365-2672.1998.00589.x