Materials and methods for detection of HPV nucleic acids

• US 9,885,092 B2
• Filed: 02/23/2012
• Issued: 02/06/2018
• Est. Priority Date: 02/24/2011
• Status: Active Grant

• Current Assignee: QIAGEN Gaithersburg, Inc. (Qiagen NV)
• Original Assignee: QIAGEN Gaithersburg, Inc. (Qiagen NV)
• Inventors: Lowe, Brian, Fulbright, Anna K., Nazarenko, Irina
• Primary Examiner(s): Wilder, Cynthia B
• Application Number:
  US13/403,759
• Publication Number:
  US 20120322049A1
• Time in Patent Office: 2,175 Days
• Field of Search: 456 912, 435 912
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/708 for papilloma
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin

Abstract

Provided are nucleic acids capable of hybridizing to HPV 16 and/or HPV 18 nucleic acids, in particular, mRNA encoding E2 and E6-7 gene products. Such nucleic acids are useful in methods of isolating RNA from a biological sample, methods and means for determining the presence of particular RNA splice-form variants in a biological sample, methods and means for determining the relative ratio of RNA ratios in a biological sample, methods and means for predicting the progression of precancerous cervical lesions, and methods and means for detecting disruption of genes or gene expression.

Claims

1. A method of detecting the presence of a target HPV RNA, the method comprising:

a) providing at least one DNA capture probe;

b) providing a first anti-RNA;

DNA hybrid antibody, wherein the first anti-RNA;

DNA hybrid antibody is bound to a support;

c) hybridizing the target HPV RNA to said at least one DNA capture probe, yielding a target RNA;

DNA capture probe complex;

d) incubating said target HPV RNA;

DNA capture probe complex with said anti-RNA;

DNA hybrid antibody, yielding a bound target HPV RNA;

DNA capture probe complex;

e) providing at least one DNA amplification probe, and hybridizing said at least one DNA amplification probe to said bound target HPV RNA;

DNA capture probe complex, yielding a bound target HPV RNA;

DNA capture/amplification probe complex;

f) providing a second anti-RNA;

DNA hybrid antibody, and incubating said bound target HPV RNA;

DNA capture/amplification probe complex with said second anti-RNA;

DNA hybrid antibody, yielding a bound target HPV RNA;

DNA;

antibody complex;

g) detecting said second anti-RNA;

DNA hybrid antibody, wherein said detecting indicates the presence of said target HPV RNA,

wherein at least one of the capture probes comprises an isolated nucleic acid having an overall length of not more than 50 nucleotides, wherein the isolated nucleic acid comprises at least one nucleotide sequence having at least 75-percent homology to a nucleotide sequence selected from the group consisting of SEQ ID NO;

1 to SEQ ID NO;

20 and SEQ ID NO;

300 to SEQ ID NO;

308, RNA equivalents thereof, and full complements thereof;

wherein the capture probe is not capable of hybridizing to more than one type of HPV genome;

wherein the amplification probe comprises at least one nucleotide sequence having at least 75-percent homology to a nucleotide sequence selected from the group consisting of SEQ ID NO;

21 to SEQ ID NO;

105 and SEQ ID NO;

111 to SEQ ID NO;

299; and

wherein the target HPV RNA is not amplified.

2. The method of claim 1, wherein the target HPV RNA is a splice variant, and wherein the at least one DNA capture probe and the at least one DNA amplification probe are selected to detect the presence of said splice variant.

3. A method for determining whether a target nucleic acid is absent from or disrupted in a sample, said method comprising:

a) treating a first portion of the sample under conditions sufficient to induce the formation of;

i) a first set of DNA;

RNA hybrids comprising the target nucleic acid; and

ii) a second set of DNA;

RNA hybrids comprising a reference nucleic acid;

b) treating a second portion of the sample under conditions sufficient to induce the formation of the second set of DNA;

RNA hybrids, but not the first set of DNA;

RNA hybrids;

c) generating a detectable signal in the first portion of the sample and the second portion of the sample, wherein the detectable signal has an intensity that correlates with the concentration of DNA;

RNA hybrids; and

d) comparing the intensity of the detectable signal in the first portion of the sample and the intensity of the detectable signal in the second portion of the sample, wherein;

i) the target nucleic acid is intact and present in the sample if the intensity of the detectable signal in the first portion of the sample is greater than the intensity of the detectable signal in the second portion of the sample; and

ii) the target nucleic acid is absent from the sample if the intensity of the detectable signal in the first portion of the sample is less than or equal to the intensity of the detectable signal in the second portion of the sample;

wherein the first portion of the sample and the second portion of the sample are formed by a method comprising contacting the sample with a first capture probe set specific for the target nucleic acid, wherein hybridization of the first capture probe set to the target nucleic acid generates a first capture complex; and

a second capture probe set specific for the reference nucleic acid, wherein hybridization of the second capture probe set to the reference nucleic acid generates a second capture complex;

wherein the first capture probe set comprises at least two capture probes having an overall length of not more than 50 nucleotides, wherein each capture probe comprises at least one nucleotide sequence having at least 75-percent homology to a nucleotide sequence selected from the group consisting of SEQ ID NO;

51, SEQ ID NO;

52, SEQ ID NO;

56 to SEQ ID NO;

59, and SEQ ID NO;

111 to SEQ ID NO;

299, RNA equivalents thereof, and full complements thereof;

wherein the second capture probe set comprises at least two capture probes having an overall length of not more than 50 nucleotides, wherein each capture probe comprises at least one nucleotide sequence having at least 75-percent homology to a nucleotide sequence selected from the group consisting of SEQ ID NO;

51, SEQ ID NO;

52, SEQ ID NO;

56 to SEQ ID NO;

59, SEQ ID NO;

111 to SEQ ID NO;

174, SEQ ID NO;

214 to SEQ ID NO;

299, RNA equivalents thereof, and full complements thereof;

wherein the first capture probe comprises;

i) a region capable of hybridizing to the target nucleic acid; and

ii) a region capable of hybridizing to a first nucleic acid sequence of an anchor probe; and

the second capture probe comprises;

i) a region capable of hybridizing to the target nucleic acid; and

ii) a region capable of hybridizing to a second nucleic acid sequence of an anchor probe, and

wherein the anchor probe is bound to or adapted to be bound to a first support and/or second support.

4. The method of claim 3, further comprising capturing the first capture complex and the second capture complex to a support.

5. The method of claim 4, wherein the first and second capture probes are bound to or adapted to be bound to the support.

6. The method of claim 5, wherein the capture probes comprise a ligand and the support comprises a ligand-binding moiety.

7. The method of claim 6, wherein the first capture probe and the second capture probe comprise the same ligand.

8. The method of claim 6, wherein the first capture probe and the second capture probe comprise different ligands, wherein:

a) the first portion of the sample is contacted with a first set of solid supports comprising a ligand binding moiety capable of binding the ligand of the first capture probe and a ligand binding moiety capable of binding the ligand of the second capture probe; and

b) the second portion of the sample is contacted with a second set of solid supports comprising a ligand binding moiety capable of binding the ligand of the second capture probe, but not the first capture probe.

9. The method of claim 8, wherein the first portion of the sample and the second portion of the sample is contacted with

i) a first probe set comprising a plurality of signal probes capable of hybridizing to the target nucleic acid;

and

ii) a second probe set comprising a plurality of signal probes capable of hybridizing to the reference nucleic acid.

10. The method of claim 6, wherein the first capture probe and the second capture probe are biotinylated and wherein the first support and the second support comprise a biotin-binding moiety.

11. The method of claim 6, wherein the capture probes are covalently bound to the support.

12. The method of claim 4, wherein:

a) the first and second capture complexes comprise a DNA;

RNA hybrid; and

b) the first and second capture complexes are captured to the first and second supports by a method comprising contacting the first and second capture complexes with an entity capable of specifically binding to a DNA;

RNA hybrid, wherein the entity capable of specifically binding to a DNA;

RNA hybrid is bound to the support or adapted to be bound to the support.

13. The method of claim 12, wherein the entity capable of specifically binding to a DNA:

RNA hybrid comprises a ligand and the first and second supports comprise a ligand-binding moiety.

14. The method of claim 13, wherein the entity capable of specifically binding to a DNA:

RNA hybrid is biotinylated and wherein the support comprises a biotin-binding moiety.

15. The method of claim 12, wherein the entity capable of specifically binding to a DNA:

RNA hybrid is covalently bound to the support.

16. The method of claim 12, wherein the entity capable of specifically binding to a DNA:

RNA hybrid is a DNA;

RNA hybrid-specific antibody or a fragment thereof.

17. The method of claim 3, wherein the first nucleic acid sequence and the second nucleic acid sequence are the same.

18. The method of claim 3, wherein the first nucleic acid sequence and the second nucleic acid sequence are different.

19. The method of claim 18, wherein the first nucleic acid sequence and the second nucleic acid sequence are disposed in the same anchor probe.

20. The method of claim 18, wherein the first nucleic acid sequence and the second nucleic acid sequence are disposed in different anchor probes.

21. The method of claim 3, wherein:

a) the first support comprises an anchor probe comprising the first nucleic acid sequence and an anchor probe comprising second nucleic acid sequence; and

b) the second support comprises anchor probes comprising the second nucleic acid sequence, but does not comprise anchor probes comprising the first nucleic acid sequence.

22. The method of claim 3, wherein:

a) the first set of DNA;

RNA hybrids is formed by a method comprising contacting the sample with a first signal probe capable of hybridizing to the target nucleic acid; and

b) the second set of DNA;

RNA hybrids is formed by a method comprising contacting the sample with a second signal probe capable of hybridizing to the reference nucleic acid.

23. The method of claim 22, wherein:

a) the first portion of the sample is contacted with the first signal probe and the second signal probe; and

b) the second portion of the sample is contacted with the second signal probe, but not the first signal probe,

wherein the first signal probe is specific for the target nucleic acid and the second signal probe is specific for the reference nucleic acid.

24. The method of claim 13, wherein:

a) the first signal probe is disposed in a first probe set comprising a plurality of signal probes capable of hybridizing to the target nucleic acid; and

b) the second signal probe is disposed in a second probe set comprising a plurality of signal probes capable of hybridizing to the reference nucleic acid.

25. The method of claim 24, wherein:

a) the plurality of signal probes of the first probe set is capable of hybridizing to at least 70% of the target nucleic acid;

b) the plurality of signal probes of the second probe set is capable of hybridizing to at least 70% of the reference nucleic acid.

26. The method of claim 3, wherein the detectable signal is generated by a method comprising contacting the first portion of the sample and the second portion of the sample with an entity capable of specifically binding to a DNA:

RNA hybrid.

27. The method of claim 26, wherein the entity capable of specifically binding a DNA:

RNA hybrid is an DNA;

RNA hybrid-specific antibody or a fragment thereof.

28. The method of claim 3, further comprising:

a) generating the first portion of the sample and the second portion of the sample by a method comprising;

i) contacting the sample with at least a first biotinylated capture probe specific for the target nucleic acid;

ii) contacting the sample with at least a second biotinylated capture probe specific for the reference nucleic acid;

iii) contacting the sample with a streptavidin-coated magnetic bead under conditions sufficient to permit binding of the biotinylated capture probes to the streptavidin coated bead; and

iv) separating the streptavidin coated beads into separate containers to form the first portion of the sample and the second portion of the sample;

b) forming the first set of DNA;

RNA hybrids and the second set of DNA;

RNA hybrids in the first portion of the sample by a method comprising contacting the first portion of the sample with a probe cocktail comprising;

i) a plurality of detectably labeled nucleic acid probes capable of hybridizing to the target nucleic acid, wherein said plurality is sufficient to cover the target nucleic acid; and

ii) a plurality of detectably labeled nucleic acid probes capable of hybridizing to the reference nucleic acid, wherein said plurality is sufficient to cover the target nucleic acid; and

c) forming the second set of DNA;

RNA hybrids in the second portion of the sample by a method comprising contacting the second portion of the sample with a probe cocktail comprising a plurality of detectably labeled signal probes capable of hybridizing to the reference nucleic acid, wherein said plurality is sufficient to cover the target nucleic acid,

wherein the detectable signal is generated by the detectably labeled signal probes.

29. The method of claim 3, wherein:

a) the target nucleic acid is an HPV E2 nucleic acid; and

b) the reference nucleic acid is selected from the group consisting of;

i) HPV E1 nucleic acid

ii) HPV E6/E7 nucleic acid

iii) HPV L1 nucleic acid

iv) HPV L2 nucleic acid.

30. The method of claim 29, wherein a group of reference nucleic acids are detected, the group comprising at least two reference nucleic acids selected from the group consisting of:

i) HPV E1 mRNA or cDNA

ii) HPV E6/E7 mRNA or cDNA

iii) HPV L1 mRNA or cDNA; and

iv) HPV L2 mRNA or cDNA.

31. The method of claim 30, wherein the group of reference nucleic acids comprises:

i) HPV E1 mRNA;

ii) HPV E6/E7 mRNA;

iii) HPV L1 mRNA; and

iv) HPV L2 mRNA.

32. A method of predicting the onset of HPV-induced cell transformation in a patient, said method comprising detecting the presence or absence of an HPV E2 mRNA in an HPV-infected tissue derived from the patient by the method of claim 29, wherein the absence of HPV E2 mRNA is indicative of the onset of HPV-induced cell transformation.

33. A method of detecting integration of an HPV genome into a genome of a host cell, said method comprising detecting the presence or absence of an HPV E2 mRNA in an HPV-infected tissue derived from the patient by the method of claim 29, wherein the absence of HPV E2 mRNA is indicative of integration of the HPV genome into a genome of a host cell.