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US 2005/0032090 A1Published application
Detection of Giardia
AssigneeMacquarie Research Ltd.
InventorsMatthias Dorsch, Duncan Veal
Life of this patent
Estimating the term…
Abstract
Oligonucleotide molecules for the detection of Giardia lamblia (G. lamblia which molecules hybridise under medium to high stringency conditions to unique 18S rDNA/rRNA sequences of G. lamblia, and methods for the detection of the presence of viable cells of G. lamblia in samples using the oligonucleotide molecules.
Claims18
1:
- An oligonucleotide molecule for the detection of Giardia lamblia (G. lamblia), wherein the oligonucleotide molecule hybridizes under medium to high stringency conditions to unique 18S rDNA/rRNA sequences of G. lamblia.
2:
- The oligonucleotide molecule according to claim 1 which hybridizes to unique 18S rDNA/rRNA sequences of G. lamblia under conditions of high stringency.
3:
- The oligonucleotide molecule according to claim 1 selected from the group consisting of oligonucleotides having the following nucleotide sequences:
- GCG TCC CGG GTG AGC GGG;
- (SEQ ID NO:
- 1)
- GCC CGC GGG CGC CCG CCC;
- (SEQ ID NO:
- 2)
- TGG GCC CGC CTC GCT CGC;
- (SEQ ID NO:
- 3)
- CGG CGG GGG GCC AAC TAC;
- (SEQ ID NO:
- 4)
- GCG GGT CCA ACG GGC CTG;
- (SEQ ID NO:
- 5)
- CGG GGC TGC CGC GGC GCG;
- (SEQ ID NO:
- 6)
- oligonucleotides comprising a part of the sequences above having at least ten bases which hybridize to unique rDNA/rRNA sequences of G. lamblia.
17 more elements
4:
- The oligonucleotide molecule according to claim 3 comprising the nucleotide sequence:
- CGG CGG GGG GCC AAC TAC.
- (SEQ ID NO:
- 4)
1 more element
5:
- The oligonucleotide molecule according to claim 3 comprising the nucleotide sequence:
- CGG GGC TGC CGC GGC GCG.
- (SEQ ID NO:
- 6)
1 more element
6:
- The oligonucleotide molecule according to claim 1 being detectably labelled.
7:
- The oligonucleotide molecule according to claim 6 wherein the label is selected from the group consisting of fluorochrome, radioisotope, and chemical.
8:
- The oligonucleotide molecule according to claim 7 wherein the label is a fluorochrome.
9:
- The oligonucleotide molecule according to claim 7 wherein the fluorochrome is selected from the group consisting of fluorescein isothiocyanate (FITC, green), cyanine dyes Cy2, Cy3, Cy3.5, Cy5, Cy5.5 that range from green to far red), and Texas Red.
10:
- A method for the detection of the presence of viable cells of G. lamblia in a sample, the method comprising the steps of:
- (a) adding to the sample an effective amount of a probe consisting of a detectably labelled oligonucleotide molecule which hybridizes under medium to high stringency conditions to unique 18S rDNA/rRNA sequences of G. lamblia;
- (b) creating and maintaining conditions effective for hybridization of the probe to the 18S rDNA/rRNA of any G. lamblia cells present in the sample;
- and
- (c) detecting hybridization of the probe and G. lambia nucleotides.
2 more elements
11:
- The method according to claim 10 wherein the probe is selected from the group consisting of oligonucleotides having one or more of the following nucleotide sequences:
- GCG TCC CGG GTG AGC GGG
- (SEQ ID NO:
- 1)
- GCC CGC GGG CGC CCG CCC
- (SEQ ID NO:
- 2)
- TGG GCC CGC CTC GCT CGC
- (SEQ ID NO:
- 3)
- CGG CGG GGG GCC AAC TAC
- (SEQ ID NO:
- 4)
- GCG GGT CCA ACG GGC CTG
- (SEQ ID NO:
- 5)
- CGG GGC TGC CGC GGC GCG
- (SEQ ID NO:
- 6)
- and oligonucleotides comprising a part of the sequences above having at least ten bases which hybridize to unique rDNA/rRNA sequences of G. lamblia.
17 more elements
12:
- The method according to claim 11 wherein the oligonucleotide molecule comprises the nucleotide sequence:
- CGG CGG GGG GCC AAC TAC.
- (SEQ ID NO:
- 4)
1 more element
13:
- The method according to claim 11 wherein the oligonucleotide molecule comprises the nucleotide sequence:
- CGG GGC TGC CGC GGC GCG.
- (SEQ ID NO:
- 6)
1 more element
14:
- The method according to claim 10 comprising detection via fluorescence in situ hybridization (FISH) in which the oligonucleotide probe is labelled with fluorochrome and after hybridization, the resulting fluorescent-labelled cell is detected by epifluorescence microscopy or flow cytometry.
15:
- The method according to claim 10 wherein at least two different oligonucleotide probes are used and are distinguished by the use of different labels on each probe.
16:
- The method according to claim 15 wherein the oligonucleotide probes are labelled with different fluorochromes and are detected by flow cytometry.
17:
- The method according to claim 10 wherein the sample is an environmental sample.
18:
- An oligonucleotide molecule which hybridizes to G. lamblia 18S rDNA/rRNA sequences under medium to high stringency conditions, wherein the oligonucleotide molecule hybridizes to at least one of target regions of G. lamblia rDNA having the following nucleotide sequences:
- CCC GCT CAC CCG GGA CGC;
- (SEQ ID NO:
- 7)
- GGG CGG GCG CCC GCG GGC;
- (SEQ ID NO:
- 8)
- GCG AGC GAG GCG GGC CCA;
- (SEQ ID NO:
- 9)
- GTA GTT GGC CCC CCG CCG;
- (SEQ ID NO:
- 10)
- CAG GCC CGT TGG ACC CGC;
- (SEQ ID NO:
- 11)
- CGC GCC GCG GCA GCC CCG.
- (SEQ ID NO:
- 12)
16 more elements