Claims
- 1. An isolated oligonucleotide not more than 60 nucleotides in length comprising:
(a) a nucleotide sequence of at least 10 contiguous nucleotides from a sequence selected from the group consisting of SEQ ID NOs: 1-10, 13, 14, and 15; (b) a nucleotide sequence having 90% sequence identity to a nucleotide sequence of (a); or (c) complements of (a) and (b).
- 2. The oligonucleotide of claim 1, wherein the oligonucleotide is a nucleotide sequence of at least 10 contiguous nucleotides from a sequence selected from the group consisting of SEQ ID NOs: 1-10, 13, 14, and 15.
- 3. The oligonucleotide of claim 1, wherein the nucleotide sequence comprises SEQ ID NO: 1.
- 4. The oligonucleotide of claim 1, wherein the nucleotide sequence comprises SEQ ID NO: 2.
- 5. The oligonucleotide of claim 1, wherein the nucleotide sequence comprises SEQ ID NO: 3.
- 6. The oligonucleotide of claim 1, wherein the nucleotide sequence comprises SEQ ID NO: 4.
- 7. The oligonucleotide of claim 1, wherein the nucleotide sequence comprises SEQ ID NO: 5.
- 8. The oligonucleotide of claim 1, wherein the nucleotide sequence comprises SEQ ID NO: 6.
- 9. The oligonucleotide of claim 1, wherein the nucleotide sequence comprises SEQ ID NO: 7.
- 10. The oligonucleotide of claim 9, further comprising a detectable label at the 5′-end and/or the 3′-end.
- 11. The oligonucleotide of claim 10, wherein the detectable label is a fluorescent label selected from the group consisting of 6-carboxyfluorescein (6-FAM), tetramethyl rhodamine (TAMRA), and 2′,4′,5′,7′,-tetrachloro-4-7-dichlorofluorescein (TET).
- 12. The oligonucleotide of claim 1, wherein the nucleotide sequence comprises SEQ ID NO: 8.
- 13. The oligonucleotide of claim 1, wherein the nucleotide sequence comprises SEQ ID NO: 9.
- 14. The oligonucleotide of claim 1, wherein the nucleotide sequence comprises SEQ ID NO: 10.
- 15. The oligonucleotide of claim 1, wherein the nucleotide sequence comprises SEQ ID NO: 13.
- 16. The oligonucleotide of claim 15, further comprising a detectable label at the 5′-end and/or the 3′-end.
- 17. The oligonucleotide of claim 16, wherein the detectable label is a fluorescent label selected from the group consisting of 6-carboxyfluorescein (6-FAM), tetramethyl rhodamine (TAMRA), and 2′,4′,5′,7′,-tetrachloro-4-7-dichlorofluorescein (TET).
- 18. The oligonucleotide of claim 1, wherein the nucleotide sequence comprises SEQ ID NO: 14.
- 19. The oligonucleotide of claim 18, further comprising a detectable label at the 5′-end and/or the 3′-end.
- 20. The oligonucleotide of claim 19, wherein the detectable label is a fluorescent label selected from the group consisting of 6-carboxyfluorescein (6-FAM), tetramethyl rhodamine (TAMRA), and 2′,4′,5′,7′,-tetrachloro-4-7-dichlorofluorescein (TET).
- 21. A method for detecting Hepatitis B virus (HBV) infection in a biological sample, the method comprising:
isolating nucleic acids from a biological sample suspected of containing HBV; amplifying the nucleic acids using at least two primers wherein (a) each of the primers is not more than about 60 nucleotides in length and comprises a nucleotide sequence of at least 10 contiguous nucleotides from a sequence selected from the group consisting of SEQ ID NOs: 5, 6, 8, 9, and 10 or (b) primers having 90% sequence identity to a nucleotide sequence of (a), wherein each of the two primers is sufficiently complementary to a portion of the sense and antisense strands, respectively, of the isolated nucleic acid to hybridize therewith; and detecting the presence of the amplified nucleic acids as an indication of the presence or absence of HBV in the sample.
- 22. The method of claim 21, wherein the nucleic acids are isolated from the biological sample by a method comprising:
(a) contacting a solid support comprising capture nucleic acids associated therewith with a biological sample under hybridizing conditions wherein target nucleic acid strands hybridize with the capture nucleic acids; and (b) separating the solid support from the sample.
- 23. The method of claim 22, wherein the solid support comprises beads.
- 24. The method of claim 23, wherein the beads are magnetic beads.
- 25. The method of claim 22, wherein the capture nucleic acids comprise one or more oligonucleotides, wherein each of the oligonculeotides is not more than about 60 nucleotides in length and comprises at least 10 contiguous nucleotides from a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4.
- 26. The method of claim 25, wherein the capture nucleic acids further comprise a homopolymer chain of about 15-25 nucleotides in length, selected from the group consisting of polyA, polyT, polyG, polyC, and polyU.
- 27. The method of claim 26, wherein the homopolymer chain is a polyA chain.
- 28. The method of claim 21, wherein amplifying comprises PCR, transcription-mediated amplification (TMA) or TaqMan.
- 29. The method of claim 28, wherein amplifying comprises TMA.
- 30. The method of claim 29, wherein the sense primer is an oligonucleotide of not more than 60 nucleotides in length comprising SEQ ID NO: 5.
- 31. The method of claim 29, wherein the antisense primers are two oligonucleotides, wherein each of the oligonucleotides is not more than about 60 nucleotides in length and comprises at least 10 contiguous nucleotides of sequences comprising SEQ ID NOs: 6 and 8 or SEQ ID NOs: 9 and 10.
- 32. The method of claim 29, further comprising a probe oligonucleotide of not more than about 60 nucleotides in length and at least 10 contiguous nucleotides comprising SEQ ID NO: 7.
- 33. The method of claim 32, wherein the probe further comprises detectable labels at the 5′-end and at the 3′-end.
- 34. The oligonucleotide of claim 33, wherein the detectable label is a fluorescent label selected from the group consisting of 6-carboxyfluorescein (6-FAM), tetramethyl rhodamine (TAMRA), and 2′,4′,5′,7′,-tetrachloro-4-7-dichlorofluorescein (TET).
- 35. The method of claim 21, further comprising an internal standard.
- 36. The method of claim 35, wherein the internal standard comprises a labeled oligonucleotide of not more than about 60 nucleotides in length and comprises a nucleotide sequence of at least 10 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 13 and 14.
- 37. The method of claim 36, wherein the label is a fluorescent label selected from the group consisting of 6-carboxyfluorescein (6-FAM), tetramethyl rhodamine (TAMRA), and 2′,4′,5′,7′,-tetrachloro-4-7-dichlorofluorescein (TET).
- 38. A method for detecting Hepatitis B virus (HBV) infection in a biological sample, the method comprising:
(a) contacting a solid support with one or more capture nucleic acids selected from the group consisting of SEQ ID NOs: 1, 2, 3, and 4 under conditions wherein the capture nucleic acids become associated with the solid support, (b) contacting the solid support of (a) with the biological sample under hybridizing conditions wherein target nucleic acid strands from HBV when present hybridize with the capture nucleic acids; and (c) separating the solid support of (b) from the sample; (d) amplifying the nucleic acids using a sense primer comprising SEQ ID NO:5 and at least two antisense primers comprising SEQ ID NOs: 6 and 8 or SEQ ID Nos: 9 and 10, wherein the sense and antisense primers are sufficiently complementary to a portion of the sense and antisense strands, respectively, of the isolated nucleic acid to hybridize therewith; and (e) detecting the presence of the amplified nucleic acids as an indication of the presence or absence of HBV in the sample.
- 39. The method of claim 38, wherein the solid support comprises beads.
- 40. The method of claim 39, wherein the beads are magnetic beads.
- 41. The method of claim 38, further comprising an internal standard.
- 42. The method of claim 41, wherein the internal standard comprises a labeled oligonucleotide of not more than about 60 nucleotides in length and comprises a nucleotide sequence of at least 10 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 13 and 14.
- 43. The method of claim 42, wherein the label is a fluorescent label selected from the group consisting of 6-carboxyfluorescein (6-FAM), tetramethyl rhodamine (TAMRA), and 2′,4′,5′,7′,-tetrachloro-4-7-dichlorofluorescein (TET).
- 44. Isolated oligonucleotides for use in capturing HBV nucleic acids comprising one or more oligonucleotides, wherein each of the oligonucleotides is not more than about 60 nucleotides in length and comprises a nucleotide sequence of at least 10 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4.
- 45. The oligonucleotides of claim 43, wherein one or more oligonucleotides further comprise a homopolymer chain of about 15-25 nucleotides in length, selected from the group consisting of polyA, polyT, polyG, polyC, and polyU.
- 46. The oligonucleotides of claim 45, wherein the homopolymer chain is a polyA chain.
- 47. Primers for use in detecting HBV nucleic acids comprising sense and antisense primers, wherein the sense and the antisense primers are not more than about 60 nucleotides in length and comprise a nucleotide sequence of at least 10 contiguous nucleotides wherein the sense primer comprises SEQ ID NO:5 and the antisense primers are selected from the group consisting of SEQ ID NOs: 6, 8, 9 and 10.
- 48. The primers of claim 47, wherein the antisense primers consist of SEQ ID NOs: 9 and 10, respectively.
- 49. A kit for detecting Hepatitis B virus (HBV) infection in a biological sample, the kit comprising:
capture nucleic acids comprising one or more oligonucleotides, wherein each of the oligonucleotides is not more than about 60 nucleotides in length and comprises a nucleotide sequence of at least 10 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4.; primer oligonucleotides wherein the primer oligonucleotides are not more than about 60 nucleotides in length and comprise a nucleotide sequence of at least 10 contiguous nucleotides from a sequence selected from the group consisting of SEQ ID NOs: 5, 6, 8, 9, and 10; and written instructions for identifying HBV infection.
- 50. The kit of claim 49, further comprising a polymerase and buffers.
- 51. The kit of claim 50, further comprising an internal standard comprising a labeled oligonucleotide of not more than about 60 nucleotides in length and that comprises a nucleotide sequence of at least 10 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 13 and 14.
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is related to provisional patent applications serial No. 60/328,492 filed Oct. 9, 2001, serial No. 60/368,823 filed Mar. 29, 2002, and serial No. 60/393,561 filed Jul. 2, 2002, from which priority is claimed under 35 USC §119(e)(1) and which applications are incorporated herein by reference in their entireties.
Provisional Applications (3)
|
Number |
Date |
Country |
|
60328492 |
Oct 2001 |
US |
|
60368823 |
Mar 2002 |
US |
|
60393561 |
Jul 2002 |
US |