Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Patent
1998-03-03
2000-11-14
Arthur, Lisa B.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
435 6, 536 231, 536 243, C12Q 168, C07H 2104, C12P 1934
Patent
active
06146855&
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to a method for the detection of viable oocysts or other cells of the protozoa species Cryptosporodium parvum. More particularly the invention relates to a method for such detection utilising one or more of a number of probes capable of hybridising to specific C. parvum 18S rRNA sequences.
BACKGROUND ART
The detection of Cryptosporidium oocysts in water presently relies on the concentration of particulate matter from large volumes of water prior to staining with fluorescently labelled monoclonal antibodies. Until recently, detection and identification of fluorescently labelled oocysts required examination of the sample using epifluorescence microscopy. The tedious and labour intensive nature of this detection method,. in particular the amount of fluorescent microscopy required, limited the monitoring work which could be performed. The development of flow cytometric assisted detection methods has alleviated some of these problems and enabled the routine monitoring of water for the presence of Cryptosporidium oocysts (Vesey et al., 1994A). However, a major limitation of all these methodologies is the lack of oocyst viability measurements. Methods to determine viability such as animal infectivity and excystation, are impractical because of the low number of oocysts normally present in water samples and the tedious nature of such tests.
The presence of dead Cryptosporidium oocysts in drinking water is of little significance to public health, however if oocysts are viable the risk to public health is enormous. Moreover, of the seven species of Cryptosporidium; C. parvum, C. muris, C. meleagridis, C. serpentis, C. nasorum, C. wrairi and C. baileyi only one, C. parvum is infectious to humans (Rose et al., 1988), and yet all the currently available monoclonal antibodies are not species specific. There is therefore, an urgent requirement to develop an effective method for determining the viability of C. parvum oocysts in water.
Fluorescence in situ hybridisation (FISH) is a relatively new method by which microorganisms can be specifically labelled. The technique is reliant upon the identification of a specific sequence of nucleic acid within the target organism. Probes targeting a specific nucleic acid sequence are then synthesised and labelled with a fluorochrome. The cell is then permeabilised and the complementary sequence allowed to hybridise with the target sequence resulting in specific labelling of the target cell.
The use of ribosomal RNA (rRNA) targeted oligonucleotide probes with FISH and flow cytometry has been reported by Amann et al. (1990B). Molecules of rRNA are ideal targets for fluorescently labelled nucleic probes for several reasons: (1) high sensitivity can be achieved since the target molecules are present in very high numbers; (2) a denaturation step is not required during the procedure as the target region is single stranded; and (3) rRNA has a short half life and will only be present in a high copy number in viable cells.
DISCLOSURE OF THE INVENTION
In a first aspect, the present invention provides an oligonucleotide molecule for the detection of viable cells of Cryptosporidium parvum (C. parvum), the oligonucleotide molecule characterised in that it specifically hybridises to unique 18S rRNA sequences of C. parvum.
Preferably, the oligonucleotide molecule specifically hybridises to unique 18S rRNA sequences of C. parvum under medium to high stringency conditions (Sambrook et al., 1989.), however, in most cases, conditions of high stringency will be required to ensure specific hybridisation to unique C. parvum 18S rRNA sequences.
In a preferred embodiment of the invention, the oligonucleotide molecule is selected from the group comprising oligonucleotides that hyloridize with one or more of the following nucleotide sequences: sequences (a) to (d) above specific hybridisation to unique 18S rRNA sequences of C. parvum.
Preferably, the oligonucleotide molecules according to the invention are detectably labelled so that they may be util
REFERENCES:
Kilani, R. and W. Wenman, "Geographical Variation in 18S rRNA gene sequence of Cryptospridium Parvum", International Journal for Parasitology, 303-306, 1994.
Cai, J et al., "PCR cloning and nucleotide sequence determination of the 18S rRNA genes and ITS1 of protozoan parasites Cryptosporidium parvum and Cryptosporidium muris", Biochim. Biophys. Acta, 317-320, 1992.
Vessey et al, "The use of a rRNA targeted oligonucleotide probe for fluorescent labelling of vialbe C. parvum oocysts", J. of Applied Microbiology, 429-440, 1998.
Champliaud et al. "Failure to Differentiate C. parvum from C. meleagridi based on PCR amplification of eight DNA sequences" Applied Environmental Microbiology, 1454-1458, Apr. 1998.
Morgan et al. "Differntiation between Human and Animal isolates of C. parvum using rDNA sequencing and direct PCR analysis", J. Parasitology, 825-830, 1997.
Ashbolt Nicholas John
Dorsch Matthias
Veal Duncan
Vesey Graham
Williams Keith Leslie
Arthur Lisa B.
Australian Water Technologies Pty. Ltd.
Enewold Jeanine
Macquarie Research Ltd.
Martin Alice O.
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