Compositions and methods for detecting the presence of
View Patent ↗The present invention describes novel oligonucleotides targeted to nucleic acid sequences derived from Cryptosporidium organisms, and Cryptosporidium parvum organisms in particular, which are useful for determining the presence of Cryptosporidium organisms in a test sample. The oligonucleotides of the present invention include hybridization assay probes, helper probes and amplification primers. The present invention further describes a novel method for obtaining purified ribonucleic acid from viable oocysts.
1. A hybridization assay probe comprising a detectable label and the base sequence of SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:14 or SEQ ID NO:18, wherein the probe hybridizes to nucleic acid derived from Cryptosporidium parvum under stringent conditions to form a probe:target hybrid stable for detection, and wherein the probe does not hybridize to nucleic acid derived from Cryptosporidium muris, Cryptosporidium baileyi , or Cryptosporidium wrairi to form a probe:non-target hybrid stable for detection under the stringent conditions.
2. The probe of claim 1 , wherein the probe is up to 27 bases in length.
3. The probe of claim 1 , wherein the base sequence of the probe consists of the base sequence of SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:14 or SEQ ID NO:18.
4. The probe of claim 1 , wherein the probe comprises at least one base sequence that does not stably hybridize to nucleic acid derived from Cryptosporidium parvum, Cryptosporidium muris, Cryptosporidium baileyi , or Cryptosporidium wrairi under the stringent conditions.
5. The probe of claim 4 , wherein the probe comprises a pair of base sequences that hybridize to each other when the probe is not hybridized to nucleic acid derived from Cryptosporidium parvum under the stringent conditions.
6. The probe of claim 5 , wherein the probe comprises a group of interacting labels.
7. The probe of claim 6 , wherein the interacting labels include a luminescent label and a quencher label.
8. The probe of claim 1 , wherein the probe includes at least one ribonucleotide modified to include a 2′-O-methyl substitution to the ribofuranosyl moiety.
9. The probe of claim 1 , wherein a pseudo peptide backbone joins at least a portion of the bases of the probe.
10. The probe of claim 1 , wherein the stringent conditions comprise 50 mM succinic acid, 1% (w/v) LLS, 7.5 mM aldrithiol-2, 0.6 M LiCl, 115 mM LiOH, 10 mM EDTA, 10 mM EGTA, 1.5% (v/v) ethyl alcohol (absolute), pH to 4.7, at a temperature of about 60° C.
11. A method for determining the presence of Cryptosporidium parvum in a test sample, the method comprising the steps of:
contacting the test sample with the probe of claim 1 under stringent conditions; and
determining whether the probe:target hybrid has formed under the stringent conditions as an indication of the presence of Cryptosporidium parvum in the test sample.
12. The method of claim 11 , wherein the probe is up to 27 bases in length.
13. The method of claim 11 , wherein the base sequence of the probe consists of the base sequence of SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:14 or SEQ ID NO:18.
14. The method of claim 11 , wherein the probe comprises at least one base sequence that does not stably hybridize to nucleic acid derived from Cryptosporidium parvum, Cryptosporidium muris, Cryptosporidium baileyi , or Cryptosporidium wrairi under the stringent conditions.
15. The method of claim 14 , wherein the probe comprises a pair of base sequences that hybridize to each other when the probe is not hybridized to nucleic acid derived from Cryptosporidium parvum under the stringent conditions.
16. The method of claim 15 , wherein the probe comprises a group of interacting labels.
17. The method of claim 16 , wherein the interacting labels include a luminescent label and a quencher label.
18. The method of claim 11 , wherein the probe includes at least one ribonucleotide modified to include a 2′-O-methyl substitution to the ribofuranosyl moiety.
19. The method of claim 11 , wherein a pseudo peptide backbone joins at least a portion of the bases of the probe.