Fast results hybrid capture assay and associated strategically truncated probes

• US 9,376,727 B2
• Filed: 05/24/2011
• Issued: 06/28/2016
• Est. Priority Date: 05/25/2010
• Status: Expired due to Fees

• Current Assignee: QIAGEN Gaithersburg, Inc. (Qiagen NV)
• Original Assignee: QIAGEN Gaithersburg, Inc. (Qiagen NV)
• Inventors: Mallonee, Richard, Qiu, Peter
• Primary Examiner(s): SITTON, JEHANNE SOUAYA
• Application Number:
  US13/114,344
• Publication Number:
  US 20120004128A1
• Time in Patent Office: 1,862 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/708 for papilloma

Abstract

Strategically truncated probes specific for high-risk HPV nucleic acids, and methods for making and using the same, are disclosed herein. The disclosed probes, and methods of use thereof, permit fast and reliable detection of human papillomavirus in clinical samples without significant cross-reaction.

Claims

1. A strategically truncated polynucleotide probe at least 1 kilobase in length and sharing 70% or more identity with a sequence selected from the group consisting of SEQ ID NO:

1 to SEQ ID NO;

17, SEQ ID NO;

104 to SEQ ID NO;

110, SEQ ID NO;

116, and full-length complements thereof, wherein;

said polynucleotide probe comprises at least one sequence having from 70% to 100% complementarity to at least 100 contiguous bases of each of L1, L2, E1, E2, E4, E6, and E7 of a HPV nucleic acid selected from the group consisting of SEQ ID NO;

1 to SEQ ID NO;

17, SEQ ID NO;

104 to SEQ ID NO;

110, SEQ ID NO;

116; and

wherein said polynucleotide probe does not contain any sequences of at least 100 contiguous bases having about 70% to 100% complementarity to at least 100 contiguous bases of SEQ ID NO;

18 to SEQ ID NO;

43 and full-length complements thereof.

2. The polynucleotide probe of claim 1, wherein said polynucleotide probe does not comprise any sequences having about 70% to 100% identity to SEQ ID NO:

44 to SEQ ID NO;

57, SEQ ID NO;

111 to SEQ ID NO;

115, and full-length complements thereof.

3. The polynucleotide probe of claim 1 comprising a sequence having about 70% to 100% identity to a sequence selected from the group consisting of SEQ ID NO:

97 to SEQ ID NO;

103, SEQ ID NO;

117, and full-length complements thereof.

4. The polynucleotide probe of claim 1 consisting essentially of a sequence selected from the group consisting of SEQ ID NO:

97 to SEQ ID NO;

103, SEQ ID NO;

117, and full-length complements thereof.

5. The polynucleotide probe of claim 1 consisting of a sequence selected from the group consisting of SEQ ID NO:

97 to SEQ ID NO;

103, SEQ ID NO;

117, and full-length complements thereof.

6. A kit comprising at least one polynucleotide probe of claim 1.

7. A method for generating a polynucleotide probe of claim 1, said method comprising:

a) providing a template nucleic acid comprising a sequence at least 1 kilobase in length sharing from about 70% to 100% identity with at least a portion of the high risk HPV nucleic acid;

b) comparing the sequence of the template nucleic acid with at least one low risk nucleic acid; and

c) truncating the template nucleic acid by amplifying, the template nucleic acid with a deletion primer to remove any regions of the template nucleic acid that share from about 70% to 100% identity over 100 contiguous base pairs with a region of the low risk HPV nucleic acid.

8. The method of claim 7, wherein the template nucleic acid is methylated before the template nucleic acid is amplified.

9. The method of claim 8 wherein the methylated template nucleic acid is selectively digested.

10. A method for determining the presence of a target nucleic acid in a sample, the method comprising:

a) hybridizing at least one polynucleotide probe according to claim 1 to the target nucleic acid molecule to form a double-stranded nucleic acid hybrid;

b) capturing the double-stranded nucleic acid hybrid on a support, wherein the support comprises a first antibody specific for a double-stranded nucleic acid hybrid; and

c) detecting the target nucleic acid molecule.

11. The method of claim 10, wherein the double stranded nucleic acid hybrid is a DNA:

RNA hybrid.

12. The method of claim 10 wherein the target nucleic acid is a high risk HPV nucleic acid.

13. The method of any of claim 10 wherein the sample is a biological sample.