HELPER OLIGONUCLEOTIDE FOR IMPROVED EFFICIENCY OF AMPLIFICATION AND DETECTION/QUANTITATION OF NUCLEIC ACIDS

• US 20180037945A1
• Filed: 07/26/2017
• Published: 02/08/2018
• Est. Priority Date: 08/02/2016
• Status: Active Grant

• Current Assignee: Roche Molecular Systems Inc (Roche Holding AG)
• Original Assignee: Roche Molecular Systems Inc (Roche Holding AG)
• Inventors: Mehta, Rochak
• Granted Patent: US 10,443,095 B2
• Time in Patent Office: Days
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 1/707 non-A, non-B Hepatitis, excluding hepatitis D
• CPC Class Codes: C12Q 2525/173 incorporating a polynucleotide run, e.g. polyAs, polyTs
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2537/162 Helper probe

Abstract

Improved methods for the detection and quantitation of a target nucleic acid in a sample using a non-extending helper oligonucleotide are described. The methods include contacting nucleic acids in a sample with amplification reagents including one or more primers, one or more non-extending helper oligonucleotides, and one or more probes. The non-extending helper oligonucleotide facilitates and increases the target nucleic acid accessibility of one or more of the primers, result in greater accumulation of amplicon production, thereby increasing the efficiency and sensitivity of the amplification assay, including amplification assays for Hepatitis C Virus (HCV), for example, HCV Genotype 5. Kits, articles of manufacture, and reaction mixtures are also provided.

Claims

1. A method for detecting and/or quantitating a target nucleic acid in a sample comprising:

(a) contacting nucleic acids in the sample with amplification reagents, the amplification reagents comprising;

at least an enzyme comprising DNA polymerase activity;

at least nucleoside triphosphate monomers or other nucleoside monomers;

at least one forward primer specific for the target nucleic acid, or at least one reverse primer specific for the target nucleic acid, or a combination thereof, for generating at least one amplicon;

at least one non-extending helper oligonucleotide, wherein;

(i) the non-extending helper oligonucleotide does not extend, (ii) the non-extending helper oligonucleotide anneals to part of the same portion of the target nucleic acid as at least one of the primers, and (iii) the non-extending helper oligonucleotide enhances the activity of at least one of the primers; and

at least one detectable probe specific for the amplicon or at least one DNA binding dye;

(b) incubating the nucleic acids with the amplification reagents for a period of time and under conditions sufficient for an amplification reaction to occur; and

(c) detecting the amplicon with the at least one detectable probe or the at least one DNA binding dye.

2. The method of claim 1, wherein the target nucleic acid is a microbial nucleic acid.

3. The method of claim 2, wherein the microbial nucleic acid is a bacterial nucleic acid.

4. The method of claim 2, wherein the microbial nucleic acid is a viral nucleic acid.

5. The method of claim 4, wherein the viral nucleic acid is a nucleic acid of Human Papilloma Virus (HPV), West Nile Virus (WNV), Human Immunodeficiency Virus (HIV), Hepatitis A Virus (HAV), Hepatitis B Virus (HBV), or Hepatitis C Virus (HCV).

6. The method of claim 5, wherein the viral nucleic acid is a nucleic acid of HCV.

7. The method of claim 6, wherein the HCV is HCV Genotype 5.

8. The method of any one of claims 1-7, wherein the at least one non-extending helper oligonucleotide comprises a poly(A) sequence at the 3′

-end of the at least one non-extending helper oligonucleotide.

9. The method of claim 8, wherein the poly(A) sequence is between 4-12 nucleotides in length.

11. The method of claim 7, wherein the at least one non-extending helper oligonucleotide comprises the sequence of SEQ ID NO:

5, or a complementary sequence thereof.

15. The method of claim 11, wherein the at least one forward primer comprises the sequence of SEQ ID NO:

1, or a complementary sequence thereof the at least one reverse primer comprises the sequence selected from the group consisting of SEQ ID NOs;

2 and 3, or a complementary sequence thereof; and

the at least one probe comprises the sequence of SEQ ID NO;

4, or a complementary sequence thereof.

10. (canceled)

12. (canceled)

13. (canceled)

14. (canceled)

16. A method for detecting and/or quantitating an HCV nucleic acid in a sample comprising:

(a) contacting nucleic acids in the sample with amplification reagents, the amplification reagents comprising;

at least an enzyme comprising DNA polymerase activity;

at least nucleoside triphosphate monomers or other nucleoside monomers;

at least one forward primer specific for the target nucleic acid, or at least one reverse primer specific for the target nucleic acid, or a combination thereof, for generating at least one amplicon;

at least one non-extending helper oligonucleotide, wherein;

(i) the non-extending helper oligonucleotide does not extend, (ii) the non-extending helper oligonucleotide anneals to part of the same portion of the target nucleic acid as at least one of the primers, and (iii) the non-extending helper oligonucleotide enhances the activity of at least one of the primers; and

at least one detectable probe specific for the amplicon or at least one DNA binding dye;

(b) incubating the nucleic acids with the amplification reagents for a period of time and under conditions sufficient for an amplification reaction to occur; and

(c) detecting the amplicon with the at least one detectable probe or the at least one DNA binding dye.

17. The method of claim 16, wherein the at least one non-extending helper oligonucleotide comprises a poly(A) sequence at the 3′

-end of the at least one non-extending helper oligonucleotide.

18. The method of claim 17, wherein the poly(A) sequence is between 4-12 nucleotides in length.

20. The method of claim 16, wherein the HCV is HCV Genotype 5.

21. The method of claim 20, wherein the at least one non-extending helper oligonucleotide comprises the sequence of SEQ ID NO:

5, or a complementary sequence thereof.

25. The method of claim 21, wherein the at least one forward primer comprises the sequence of SEQ ID NO:

1, or a complementary sequence thereof;

the at least one reverse primer comprises the sequence selected from the group consisting of SEQ ID NOs;

2 and/or 3, or a complementary sequence thereof; and

the at least one probe comprises the sequence of SEQ ID NO;

4, or a complementary sequence thereof.

19. (canceled)

22. (canceled)

23. (canceled)

24. (canceled)

26. A kit for amplifying and detecting and/or quantitating a target nucleic acid in a sample comprising amplification reagents, the amplification reagents comprising:

(a) an enzyme comprising DNA polymerase activity;

(b) nucleoside triphosphate monomers or other nucleoside monomers;

(c) at least one forward primer or at least one reverse primer specific for the target nucleic acid, or a combination thereof;

(d) at least one non-extending helper oligonucleotide, wherein;

(i) the non-extending helper oligonucleotide does not extend, (ii) the non-extending helper oligonucleotide anneals to part of the same portion of the target nucleic acid as at least one of the primers, and (iii) the non-extending helper oligonucleotide enhances the activity of at least one of the primers; and

(e) at least one detectable probe for the target nucleic acid or a DNA binding dye.

27. The kit of claim 26, wherein the target nucleic acid is a microbial nucleic acid.

28. The kit of claim 27, wherein the microbial nucleic acid is a bacterial nucleic acid.

29. The kit of claim 27, wherein the microbial nucleic acid is a viral nucleic acid.

30. The kit of claim 29, wherein the viral nucleic acid is a nucleic acid of HPV, WNV, HIV, HAV, HBV, or HCV.

31. The kit of claim 30, wherein the viral nucleic acid is a nucleic acid of HCV.

32. The kit of claim 31, wherein the HCV is HCV Genotype 5.

33. The kit of any one of claims 26-32, wherein the at least one non-extending helper oligonucleotide comprises a poly(A) sequence at the 3′

-end of the at least one non-extending helper oligonucleotide.

34. The kit of claim 33, wherein the poly(A) sequence is between 4-12 nucleotides in length.

36. The kit of claim 32, wherein the at least one non-extending helper oligonucleotide comprises the sequence of SEQ ID NO:

5, or a complementary sequence thereof.

40. The kit of claim 36, wherein the at least one forward primer comprises the sequence of SEQ ID NO:

1, or a complementary sequence thereof;

the at least one reverse primer comprises the sequence selected from the group consisting of SEQ ID NOs;

2 and 3, or a complementary sequence thereof; and

the at least one probe comprises the sequence of SEQ ID NO;

4, or a complementary sequence thereof.

35. (canceled)

37. (canceled)

38. (canceled)

39. (canceled)

41. A reaction mixture effective to amplify and detect and/or quantitate a target nucleic acid in a sample comprising amplification reagents comprising:

(a) a sample;

(b) an enzyme comprising DNA polymerase activity;

(c) nucleoside triphosphate monomers or other nucleoside monomers;

(d) at least one forward primer or at least one reverse primer specific for the target nucleic acid, or a combination thereof;

(e) at least one non-extending helper oligonucleotide, wherein;

(i) the non-extending helper oligonucleotide does not extend, (ii) the non-extending helper oligonucleotide anneals to part of the same portion of the target nucleic acid as at least one of the primers, and (iii) the non-extending helper oligonucleotide enhances the activity of at least one of the primers; and

at least one detectable probe for the target nucleic acid or a DNA binding dye.

42. The reaction mixture of claim 41, wherein the target nucleic acid is a microbial nucleic acid.

43. The reaction mixture of claim 42, wherein the microbial nucleic acid is a bacterial nucleic acid.

44. The reaction mixture of claim 42, wherein the microbial nucleic acid is a viral nucleic acid.

45. The reaction mixture of claim 44, wherein the viral nucleic acid is a nucleic acid of HPV, WNV, HIV, HAV, HBV, or HCV.

46. The reaction mixture of claim 45, wherein the viral nucleic acid is a nucleic acid of HCV.

47. The reaction mixture of claim 46, wherein the HCV is HCV Genotype 5.

48. The reaction mixture of claim 41, wherein the at least one non-extending helper oligonucleotide comprises a poly(A) sequence at the 3′

-end of the at least one non-extending helper oligonucleotide.

49. The reaction mixture of claim 48, wherein the poly(A) sequence is between 4-12 nucleotides in length.

51. The reaction mixture of claim 49, wherein the at least one non-extending helper oligonucleotide comprises the sequence of SEQ ID NO:

5, or a complementary sequence thereof.

55. The reaction mixture of claim 51, wherein the at least one forward primer comprises the sequence of SEQ ID NO:

1, or a complementary sequence thereof;

the at least one reverse primer comprises the sequence selected from the group consisting of SEQ ID NOs;

2 and 3, or a complementary sequence thereof; and

the at least one probe comprises the sequence of SEQ ID NO;

4, or a complementary sequence thereof.

50. (canceled)

52. (canceled)

53. (canceled)

54. (canceled)