Compositions, methods and kits for determining the presence ofCryptosporidium parvumorganisms in a test sample

• US 7,081,527 B2
• Filed: 09/11/2001
• Issued: 07/25/2006
• Est. Priority Date: 09/12/2000
• Status: Expired due to Fees

• Current Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Original Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Inventors: Stull, Paul D., Cunningham, Melissa M., Weisburg, William G.
• Primary Examiner(s): GOLDBERG, JEANINE ANNE
• Application Number:
  US09/954,586
• Publication Number:
  US 20020146717A1
• Time in Patent Office: 1,778 Days
• Field of Search: 533/23.4, 533/24.3, 533/24.33, 533/23.7, 533/24.32, 435/6, 435/91.2, 435/91.1
• US Class Current: 536/23.1
• CPC Class Codes: C12Q 1/6893 for protozoa
• CPC Class Codes: C12Q 1/6895 for plants, fungi or algae
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

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.

Claims

1. A set of amplification oligonucleotides for use in amplifying Cryptosporidium parvum nucleic acid, said set of amplification oligonucleotides comprising:

a first amplification oligonucleotide, wherein the base sequence of said first amplification oligonucleotide consists of a base sequence selected from the group consisting of SEQ ID NO;

46, SEQ ID NO;

52, SEQ ID NO;

58 and SEQ ID NO;

64 and, optionally, a 5′

sequence that is recognized by an RNA polymerase or which enhances initiation or elongation by an RNA polymerase; and

a second amplification oligonucleotide, wherein the base sequence of said second amplification oligonucleotide consists of a base sequence selected from the group consisting of SEQ ID NO;

47, SEQ ID NO;

53, SEQ ID NO;

59 and SEQ ID NO;

65 and, optionally, a 5′

sequence that is recognized by an RNA polymerase or which enhances initiation or elongation by an RNA polymerase,

wherein said amplification oligonucleotides are capable of amplifying a target sequence present in a target nucleic acid derived from Cryptosporidium parvum under amplification conditions.

2. The set of amplification oligonucleotides of claim 1, wherein at least one of said amplification oligonucleotides includes said 5′

sequence that is recognized by an RNA polymerase or which enhances initiation or elongation by an RNA polymerase.

3. A method for amplifying Cryptosporidium parvum nucleic acid that may be present in a sample, said method comprising:

contacting said sample with said amplification oligonucleotides of claim 1 under said amplification conditions; and

amplifying, if present, said target nucleic acid sequence.

4. The method of claim 3 further comprising:

contacting said sample with a hybridization assay probe, comprising a target binding region, wherein the base sequence of said target binding region consists of a base sequence selected from the group consisting of SEQ ID NO;

5, SEQ ID NO;

9, SEQ ID NO;

13 and SEQ ID NO;

17, wherein said probe forms a stable probe;

target hybrid with said target nucleic acid or its complement under stringent conditions, wherein said probe does not include a region in addition to said target binding region that hybridizes to said target nucleic acid or its complement under said stringent conditions, and wherein said probe does not form a stable probe;

non-target hybrid with nucleic acid derived from Cryptosporidium muris, Cryptosporidium baileyi or Cryptosporidium wrairi under said stringent conditions; and

determining whether said probe;

target hybrid has formed as an indication of the presence of Cryptosporidium parvum in said sample.

5. The method of claim 4, wherein at least one of said amplification oligonucleotides includes a 5′

sequence that is recognized by an RNA polymerase or which enhances initiation or elongation by an RNA polymerase.

6. The method of claim 4, wherein said probe comprises at least one base region that does not stably hybridize to said target nucleic acid or its complement under said stringent conditions.

7. The method of claim 4, wherein said probe further comprises a detectable label.

8. The method of claim 4, wherein said target binding region includes at least one ribonucleotide modified to include a 2′

-O-methyl substitution to the ribofuranosyl moiety or a pseudo peptide backbone which joins at least a portion of the bases of said target binding region.

9. A kit for use in detecting the presence of Cryptosporidium parvum in a sample, said kit comprising:

a first amplification oligonucleotide, wherein the base sequence of said first amplification oligonucleotide consists of a base sequence selected from the group consisting of SEQ ID NO;

46, SEQ ID NO;

52, SEQ ID NO;

58 and SEQ ID NO;

64 and, optionally, a 5′

sequence that is recognized by an RNA polymerase or which enhances initiation or elongation by an RNA polymerase;

a second amplification oligonucleotide, wherein the base sequence of said second amplification oligonucleotide consists of a base sequence selected from the group consisting of SEQ ID NO;

47, SEQ ID NO;

53, SEQ ID NO;

59 and SEQ ID NO;

65 and, optionally, a 5′

sequence that is recognized by an RNA polymerase or which enhances initiation or elongation by an RNA polymerase,

wherein said amplification oligonucleotides are capable of amplifying a target sequence present in a target nucleic acid derived from Cryptosporidium parvum under said amplification conditions; and

a hybridization assay probe comprising a target binding region, wherein the base sequence of said target binding region consists of a base sequence selected from the group consisting of SEQ ID NO;

5, SEQ ID NO;

9, SEQ ID NO;

13 and SEQ ID NO;

17, wherein said probe forms a stable probe;

target hybrid with said target nucleic acid or its complement under stringent conditions, wherein said probe does not include a region in addition to said target binding region that hybridizes to said target nucleic acid or its complement under said stringent conditions, and wherein said probe does not form a stable probe;

non-target hybrid with nucleic acid derived from Cryptosporidium muris, Cryptosporidium baileyi or Cryptosporidium wrairi under said stringent conditions.

10. The kit of claim 9, wherein at least one of said amplification oligonucleotides includes a 5′

sequence that is recognized by an RNA polymerase or which enhances initiation or elongation by an RNA polymerase.

11. The kit of claim 9, wherein said probe comprises at least one base region that does not stably hybridize to said target nucleic acid or its complement under said stringent conditions.

12. The kit of claim 9, wherein said probe further comprises a detectable label.

13. The kit of claim 9, wherein said target binding region includes at least one ribonucleotide modified to include a 2′

-O-methyl substitution to the ribofuranosyl moiety or a pseudo peptide backbone which joins at least a portion of the bases of said target binding region.