Compositions and methods for detecting or quantifying hepatitis B virus

Abstract

The present disclosure provides oligomers, compositions and kits for detecting and quantifying hepatitis B virus (HBV), including different genotypes and variants thereof, and related methods and uses. In some embodiments, the oligomer targets the P and / or S open reading frames of HBV and is configured to provide substantially equivalent quantification of different genotypes and variants of HBV.

Description

Technical Field

[0001] The present disclosure relates to compositions, kits and methods useful for detecting and quantifying hepatitis B virus nucleic acid. Background Art

[0002] Hepatitis B virus (HBV) can cause acute and chronic illness, with infected individuals at risk for cirrhosis and cancer. According to the July 2016 World Health Organization Hepatitis B Fact Sheet, an estimated 240 million individuals are infected worldwide, with more than 680,000 deaths each year from HBV-related liver disease. HBV can be transmitted sexually, during childbirth from infected mothers, and through other contact with body fluids, including shared items such as toothbrushes and razors, as well as needles, and poses an occupational hazard to healthcare workers.

[0003] HBV is a partially double-stranded DNA (dsDNA) virus. Its distribution is worldwide, with genotypes A to H, and multiple subtypes are known. Antiviral therapy can be effective against chronic HBV, but reliable and sensitive nucleic acid-based detection and quantification are complicated by genetic heterogeneity, which stems in part from the error-prone nature of HBV replication, in which new viral DNA is reverse transcribed from RNA by enzymes that lack proofreading activity. See, e.g., Huang CJ et al. (2013), "Impact of Genetic Heterogeneity in Polymerase of Hepatitis B Virus on Dynamics of Viral Load and Hepatitis B Progression," PLoS ONE 8(7): e70169, doi: 10.1371 / journal.pone.0070169. Hepatitis B is not easily distinguished from other types of hepatitis clinically, further emphasizing the importance of molecular detection methods (such as nucleic acid assays). Quantification can be used, for example, to monitor viral load before, during or after antiviral treatment, or to assess the severity of infection. However, it is extraordinary to measure HBV nucleic acid in a manner that reliably detects and quantifies multiple genotypes, in that a small amount of, for example, two amplification oligomers and a probe cannot amplify and quantify many known genotypes and subtypes in a comparable manner. And it may be particularly sensitive to false negatives of certain genotypes at low concentrations. On the other hand, the use of an increased number of oligomers may be complicated by factors that affect accuracy, sensitivity or specificity, such as an increased likelihood of interaction between oligomers, or having unexpected target sequences, differential amplification efficiency, resource competition, etc.

[0004] Therefore, there is a need for sensitive, specific and accurate detection and quantification of HBV regardless of genotype. Compositions, kits and methods are provided herein to meet this need, provide other benefits, or at least provide the public with a useful choice. Summary of the invention

[0005] Provided herein is a composition or kit comprising at least a first, a second, a third and a fourth amplification oligomer, wherein: the first amplification oligomer comprises a target hybridization sequence comprising at least 10 consecutive nucleotides of one of SEQ ID NOs: 2 or 3; the second amplification oligomer comprises a target hybridization sequence comprising at least 10 consecutive nucleotides of one of SEQ ID NOs: 20, 21 or 22; the third amplification oligomer comprises a target hybridization sequence comprising at least 10 consecutive nucleotides of one of SEQ ID NOs: 41; and the fourth amplification oligomer comprises a target hybridization sequence comprising at least 10 consecutive nucleotides of one of SEQ ID NOs: 34 or 35; wherein the target hybridization sequences of the first, second, third and fourth amplification oligomers each comprise at least about 14 consecutive nucleotides of a hepatitis B virus sequence or its complement.

[0006] A method for detecting hepatitis B virus in a sample is also provided, the method comprising: contacting the sample with at least a first, a second, a third and a fourth amplification oligomer to form a composition, performing a nucleic acid amplification reaction in the composition, producing at least a first and a second amplicons in the presence of hepatitis B virus nucleic acid, and quantifying the first and second amplicons, wherein: in the presence of the hepatitis B virus nucleic acid, the first amplicon is produced by extending the first and second amplification oligomers; in the presence of the hepatitis B virus nucleic acid, the second amplicon is produced by extending the third and fourth amplification oligomers; the first amplification oligomer comprises a target hybridization sequence, the target hybridization sequence comprises at least 10 consecutive nucleotides of one of SEQ ID NOs: 2 or 3; the second amplification oligomer comprises a target hybridization sequence, the target hybridization sequence comprises at least 10 consecutive nucleotides of one of SEQ ID NOs: 20, 21 or 22; the third amplification oligomer comprises a target hybridization sequence, the target hybridization sequence comprises at least 10 consecutive nucleotides of SEQ ID NO: 41; and the fourth amplification oligomer comprises a target hybridization sequence, the target hybridization sequence comprises SEQ ID NO: NO: at least 10 consecutive nucleotides of one of 34 or 35; wherein the target hybridization sequences of the first, second, third and fourth amplification oligomers each comprise at least about 14 consecutive nucleotides of a hepatitis B virus sequence or its complement.

[0007] In some embodiments, one or more of the first, second, third, and fourth amplification oligomers are promoter-primers. In some embodiments, the second amplification oligomer is a promoter-primer. In some embodiments, the fourth amplification oligomer is a promoter-primer. In some embodiments, one or more of the promoter-primers comprises a T7 promoter located 5' of the target hybridization sequence. In some embodiments, one or more of the promoter-primers comprises a sequence of SEQ ID NO: 8, 9, 10, 11, 12, or 13.

[0008] In some embodiments, one or more of the first, second, third, and fourth amplification oligomers comprise a non-nucleotide detectable label.

[0009] In some embodiments, the first amplification oligomer comprises a target hybridization sequence comprising at least one, two, or three of SEQ ID NOs: 4, 5, 6, or 7. In some embodiments, the second amplification oligomer comprises a target hybridization sequence comprising at least one, two, three, four, or five of SEQ ID NOs: 23, 24, 25, 26, 27, and 28. In some embodiments, the third amplification oligomer comprises a target hybridization sequence comprising at least one, two, or three of SEQ ID NOs: 42, 43, and 44. In some embodiments, the fourth amplification oligomer comprises a target hybridization sequence comprising at least one, two, or three of SEQ ID NOs: 36, 37, 38, and 39.

[0010] In some embodiments, the first amplification oligomer comprises at least 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides of SEQ ID NO: 3. In some embodiments, the first amplification oligomer comprises the sequence of SEQ ID NO: 3. In some embodiments, the first amplification oligomer comprises at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 consecutive nucleotides of SEQ ID NO: 2. In some embodiments, the first amplification oligomer comprises the sequence of SEQ ID NO: 2.

[0011] In some embodiments, the composition or kit further comprises an additional amplification oligomer different from the first amplification oligomer, which comprises at least 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 consecutive nucleotides of SEQ ID NO: 3. In some embodiments, the additional amplification oligomer comprises the sequence of SEQ ID NO:3.

[0012] In some embodiments, the second amplification oligomer comprises at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides of SEQ ID NO: 21. In some embodiments, the second amplification oligomer comprises the sequence of SEQ ID NO: 21. In some embodiments, the second amplification oligomer comprises the sequence of SEQ ID NO: 14. In some embodiments, the second amplification oligomer comprises at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 consecutive nucleotides of SEQ ID NO: 22. In some embodiments, the second amplification oligomer comprises the sequence of SEQ ID NO: 22. In some embodiments, the second amplification oligomer comprises the sequence of SEQ ID NO: 15. In some embodiments, the second amplification oligomer comprises at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 consecutive nucleotides of SEQ ID NO: 20. In some embodiments, the second amplification oligomer comprises the sequence of SEQ ID NO: 20.

[0013] In some embodiments, the third amplification oligomer comprises at least 11, 12, 13, 14, 15, 16, 17, 18, or 19 consecutive nucleotides of SEQ ID NO: 41. In some embodiments, the third amplification oligomer comprises the sequence of SEQ ID NO:41.

[0014] In some embodiments, the fourth amplification oligomer comprises at least 10 consecutive nucleotides of SEQ ID NO: 35, including inosine at position 30 of SEQ ID NO: 35. In some embodiments, the fourth amplification oligomer comprises at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 consecutive nucleotides of SEQ ID NO: 34. In some embodiments, the fourth amplification oligomer comprises the sequence of SEQ ID NO: 34. In some embodiments, the fourth amplification oligomer comprises the sequence of SEQ ID NO: 30.

[0015] In some embodiments, the composition or kit further comprises a fifth amplification oligomer comprising at least 10 consecutive nucleotides of the sequence of SEQ ID NO: 69 and at least about 14 consecutive nucleotides of a hepatitis B virus sequence or its complement. In some embodiments, the fifth amplification oligomer comprises at least one or two of SEQ ID NO: 70, 71 or 72. In some embodiments, the fifth amplification oligomer comprises at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 consecutive nucleotides of SEQ ID NO: 69. In some embodiments, the fifth amplification oligomer comprises the sequence of SEQ ID NO: 69. In some embodiments, the fifth amplification oligomer is a promoter-primer. In some embodiments, the fifth amplification oligomer comprises the sequence of SEQ ID NO: 67.

[0016] In some embodiments, the composition or kit further comprises a sixth amplification oligomer comprising at least 10 consecutive nucleotides of the sequence of SEQ ID NO: 73 and at least about 14 consecutive nucleotides of a hepatitis B virus sequence or its complement. In some embodiments, the sixth amplification oligomer comprises at least one or two of SEQ ID NO: 74, 75, or 76. In some embodiments, the sixth amplification oligomer comprises at least 11, 12, 13, 14, 15, 16, 17, 18, or 19 consecutive nucleotides of SEQ ID NO: 73. In some embodiments, the sixth amplification oligomer comprises the sequence of SEQ ID NO: 73.

[0017] In some embodiments, the composition further comprises HBV nucleic acid.

[0018] In some embodiments, the composition or kit further comprises at least one DNA polymerase. In some embodiments, the DNA polymerase is a reverse transcriptase. In some embodiments, the DNA polymerase is thermophilic. In some embodiments, the DNA polymerase is mesophilic.

[0019] In some embodiments, the composition or kit further comprises an RNA polymerase. In some embodiments, the RNA polymerase is a T7 RNA polymerase.

[0020] In some embodiments, the composition or kit further comprises at least one, at least two or each of Mg2+, buffer and dNTP. In some embodiments, the composition or kit further comprises rNTP.

[0021] In some embodiments, the composition or kit further comprises a first control amplification oligomer and a second control amplification oligomer that do not specifically hybridize to HBV. In some embodiments, the first control amplification oligomer comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 consecutive nucleotides of the SEQ ID NO: 77 sequence. In some embodiments, the second control amplification oligomer comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 consecutive nucleotides of the SEQ ID NO: 86 sequence. In some embodiments, the first control amplification oligomer or the second control amplification oligomer is a promoter-primer. In some embodiments, the composition or kit further comprises at least one control probe oligomer configured to specifically hybridize to the amplicon produced by the first and second control amplification oligomers. In some embodiments, the control probe oligomer comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides of SEQ ID NO:79.

[0022] In some embodiments, the composition or kit further comprises at least one probe oligomer configured to specifically hybridize to the amplicon produced by the first and second amplification oligomers. In some embodiments, the probe oligomer comprises at least 10 consecutive nucleotides of the sequence of SEQ ID NO: 29 and at least about 14 consecutive nucleotides of the hepatitis B virus sequence or its complement. In some embodiments, the probe oligomer comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 consecutive nucleotides of SEQ ID NO: 82. In some embodiments, the probe oligomer comprises the sequence of SEQ ID NO: 82. In some embodiments, the probe oligomer comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 consecutive nucleotides of SEQ ID NO: 82. In some embodiments, the probe oligomer comprises the sequence of SEQ ID NO:29.

[0023] In some embodiments, the composition or kit further comprises at least one probe oligomer configured to specifically hybridize with the amplicon produced by the third and fourth amplification oligomers. In some embodiments, the probe oligomer comprises at least 10 consecutive nucleotides of the sequence of SEQ ID NO: 40 and at least about 14 consecutive nucleotides of the hepatitis B virus sequence or its complement. In some embodiments, the probe oligomer comprises at least 10, 11, 12, 13, 14 or 15 consecutive nucleotides of SEQ ID NO: 83. In some embodiments, the probe oligomer comprises the sequence of SEQ ID NO: 83. In some embodiments, the probe oligomer comprises at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 consecutive nucleotides of SEQ ID NO: 40. A probe oligomer is also provided, comprising a target hybridization region, the target hybridization region comprising the sequence of SEQ ID NO: 83. In some embodiments, the probe oligomer comprises the sequence of SEQ ID NO: 84. In some embodiments, the probe oligomer comprises the sequence of SEQ ID NO: 85. In some embodiments, the probe oligomer comprises at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive nucleotides of SEQ ID NO: 40. In some embodiments, the probe oligomer comprises the sequence of SEQ ID NO: 40.

[0024] Also provided is an amplification oligomer comprising a target hybridization region, the target hybridization region comprising at least 10 consecutive nucleotides of SEQ ID NO: 35, including inosine at position 30 of SEQ ID NO: 35 and at least 14 consecutive nucleotides of the HBV sequence. In some embodiments, the amplification oligomer comprising a subsequence of SEQ ID NO: 34 or 35 comprises at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 consecutive nucleotides of SEQ ID NO: 35. In some embodiments, the amplification oligomer comprising a subsequence of SEQ ID NO: 34 or 35 comprises a sequence of SEQ ID NO: 35. In some embodiments, the amplification oligomer comprising a subsequence of SEQ ID NO: 34 or 35 comprises a sequence of SEQ ID NO: 31.

[0025] In some embodiments, at least one probe oligomer comprises a non-nucleotide detectable label. In some embodiments, the non-nucleotide detectable label is a fluorescent label. In some embodiments, the probe oligomer comprises a quencher. In some embodiments, the non-nucleotide detectable label is a fluorescent label, and the quencher absorbs fluorescence to a greater extent when the probe is free than when the probe is annealed to the target nucleic acid. In some embodiments, the fluorescent label is FAM, HEX or acridine. In some embodiments, the quencher is DABCYL or ROX. In some embodiments, the fluorescent label is attached to the 5'-end of the probe oligomer and the quencher is attached to the 3'-end of the probe oligomer, or the fluorescent label is attached to the 3'-end of the probe oligomer and the quencher is attached to the 5'-end of the probe oligomer. In some embodiments, at least about half, at least about 90% or all of the sugars in the probe oligomer are 2'-O-methyl-ribose. In some embodiments, the probe oligomer comprises a first self-complementary region at its 5' end and a second self-complementary region at its 3' end. In some embodiments, the self-complementary regions can hybridize to form about 4 to 7 Watson-Crick or wobble base pairs. In some embodiments, the self-complementary regions can hybridize to form about 5 Watson-Crick or wobble base pairs.

[0026] Also provided is a composition or kit comprising two or more different capture oligomers selected from the following capture oligomers (i)-(iv): (i) a first capture oligomer comprising a target hybridization sequence, wherein the target hybridization sequence comprises at least 10 consecutive nucleotides of SEQ ID NO: 49 or 99; (ii) a second capture oligomer comprising a target hybridization sequence, wherein the target hybridization sequence comprises at least 10 consecutive nucleotides of SEQ ID NO: 53, 100, 101 or 104; (iii) a third capture oligomer comprising a target hybridization sequence, wherein the target hybridization sequence comprises at least 10 consecutive nucleotides of SEQ ID NO: 57, 96 or 102; (iv) a fourth capture oligomer comprising a target hybridization sequence, wherein the target hybridization sequence comprises at least 10 consecutive nucleotides of SEQ ID NO: 61, 97, 98 or 103; wherein the target hybridization sequences of the two or more different capture oligomers each comprise at least about 14 consecutive nucleotides of a hepatitis B virus sequence or its complement.

[0027] A method for separating HBV nucleic acid from a sample is also provided, the method comprising: contacting the sample with two or more different capture oligomers selected from the following capture oligomers (i)-(iv) under conditions that allow formation of one or more complexes of capture oligomers and HBV nucleic acid, thereby forming a composition: (i) a first capture oligomer comprising a target hybridization sequence, the target hybridization sequence comprising at least 10 consecutive nucleotides of SEQ ID NO: 49 or 99; (ii) a second capture oligomer comprising a target hybridization sequence, the target hybridization sequence comprising at least 10 consecutive nucleotides of SEQ ID NO: 53, 100, 101 or 104; (iii) a third capture oligomer comprising a target hybridization sequence, the target hybridization sequence comprising at least 10 consecutive nucleotides of SEQ ID NO: 57, 96 or 102; (iv) a fourth capture oligomer comprising a target hybridization sequence, the target hybridization sequence comprising SEQ ID NO: NO: 61, 97, 98 or 103; wherein the target hybridization sequences of the two or more different capture oligomers each contain at least about 14 consecutive nucleotides of a hepatitis B virus sequence or its complement; and separating the capture oligomer from the composition. In some embodiments, separating the capture oligomer comprises binding the capture oligomer to a solid support and washing the solid support. In some embodiments, the solid support comprises a poly-N sequence complementary to a portion of at least one or at least two capture oligomers. In some embodiments, the solid support comprises a binding agent that recognizes an affinity tag present in at least one or at least two capture oligomers.

[0028] In some embodiments, the composition or kit comprises at least three of the capture oligomers (i)-(iv). In some embodiments, the composition or kit comprises capture oligomers (i)-(iv).

[0029] In some embodiments, the first capture oligomer comprises a target hybridization sequence comprising at least one of SEQ ID NOs: 50, 51, and 52. In some embodiments, the first capture oligomer comprises at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 consecutive nucleotides of SEQ ID NO: 49. In some embodiments, the first capture oligomer comprises the sequence of SEQ ID NO: 49.

[0030] In some embodiments, the second capture oligomer comprises a target hybridization sequence comprising at least one of SEQ ID NOs: 54, 55, and 56. In some embodiments, the second capture oligomer comprises at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 consecutive nucleotides of the sequence of SEQ ID NO: 53. In some embodiments, the second capture oligomer consists of SEQ ID NO: 53.

[0031] In some embodiments, the third capture oligomer comprises a target hybridization sequence comprising at least one of SEQ ID NOs: 58, 59, and 60. In some embodiments, the third capture oligomer comprises at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive nucleotides of SEQ ID NO: 57. In some embodiments, the third capture oligomer comprises the sequence of SEQ ID NO: 57.

[0032] In some embodiments, the fourth capture oligomer comprises a target hybridization sequence comprising at least one of SEQ ID NOs: 62, 63, and 64. In some embodiments, the fourth capture oligomer comprises at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 consecutive nucleotides of SEQ ID NO: 61. In some embodiments, the fourth capture oligomer comprises the sequence of SEQ ID NO: 61.

[0033] In some embodiments, the at least one capture oligomer further comprises a non-nucleotide affinity tag. In some embodiments, the at least one capture oligomer further comprises a non-HBV sequence. In some embodiments, the two, three or four capture oligomers further comprise a non-HBV sequence. In some embodiments, the at least one, two, three or four capture oligomers further comprise a poly-N sequence. In some embodiments, the poly-N sequence is a poly-A or poly-T sequence.

[0034] In some embodiments, the composition or kit further comprises at least one amplification oligomer selected from: a first amplification oligomer comprising a target hybridization sequence comprising at least 10 consecutive nucleotides of one of SEQ ID NOs: 2 or 3; a second amplification oligomer comprising a target hybridization sequence comprising at least 10 consecutive nucleotides of one of SEQ ID NOs: 20, 21 or 22; a third amplification oligomer comprising a target hybridization sequence comprising at least 10 consecutive nucleotides of one of SEQ ID NOs: 41; a fourth amplification oligomer comprising a target hybridization sequence comprising at least 10 consecutive nucleotides of one of SEQ ID NOs: 34 or 35; wherein the target hybridization sequences of the first, second, third and fourth amplification oligomers each comprise at least about 14 consecutive nucleotides of a hepatitis B virus sequence or its complement.

[0035] In some embodiments, the composition or kit comprises a first amplification oligomer disclosed herein. In some embodiments, the composition or kit comprises a second amplification oligomer disclosed herein. In some embodiments, the second amplification oligomer comprises at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 consecutive nucleotides of SEQ ID NO: 22, and the composition or kit further comprises an additional amplification oligomer different from the second amplification oligomer and comprises at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 consecutive nucleotides of SEQ ID NO: 20. In some embodiments, the second amplification oligomer comprises the sequence of SEQ ID NO: 22. In some embodiments, the additional amplification oligomer comprises the sequence of SEQ ID NO: 20. The composition, kit or method of any one of claims 113-118, wherein the composition or kit comprises a third amplification oligomer disclosed herein. In some embodiments, the composition or kit comprises an amplification oligomer comprising a target hybridization sequence comprising at least 10 consecutive nucleotides of one of SEQ ID NOs: 34 or 35 disclosed herein.

[0036] In some embodiments, the method further comprises implementing linear amplification, wherein at least one amplification oligomer is extended. In some embodiments, before the linear amplification, the amplification oligomer is bound to a complex of HBV nucleic acid and capture oligomer, and the complex is bound to a solid support, and the method comprises washing the solid support. In some embodiments, the solid support is a microbead population. In some embodiments, the microbeads of the population are magnetic. In some embodiments, after the washing step, the method comprises adding one or more additional amplification oligomers, the additional amplification oligomers being oriented oppositely to the amplification oligomer bound to the complex of HBV nucleic acid and the capture oligomer. In some embodiments, the one or more other amplification oligomers of opposite orientation are promoter-primers. In some embodiments, the one or more other amplification oligomers of opposite orientation are not promoter-primers. In some embodiments, the one or more other amplification oligomers of opposite orientation include the first amplification oligomer disclosed herein. In some embodiments, the one or more other amplification oligomers of opposite orientation include the second amplification oligomer disclosed herein. In some embodiments, one or more oppositely oriented additional amplification oligomers include a third amplification oligomer disclosed herein. In some embodiments, the one or more oppositely oriented additional amplification oligomers include an amplification oligomer comprising a target hybridization sequence comprising at least 10 consecutive nucleotides of one of SEQ ID NOs: 34 or 35 disclosed herein.

[0037] In some embodiments, the method further comprises performing exponential amplification after the linear amplification. In some embodiments, the exponential amplification is transcription-mediated amplification.

[0038] In some embodiments, one, two, three, four or more target hybridizing sequences of the oligomer comprise at least about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 consecutive nucleotides of a Hepatitis B virus sequence or its complement.

[0039] Also provided is a method for determining the level of hepatitis B virus in a sample, the sample comprising first and second hepatitis B amplicons bound to first and second markers, respectively, the method comprising: detecting a first signal emitted from the first marker; detecting a second signal emitted from the second marker; determining whether the first signal or the second signal is above a predetermined threshold; and calculating the level of hepatitis B virus in the sample, wherein if the first signal or the second signal is above the predetermined threshold, the level is calculated from the larger of the first and second signals; and wherein if the first signal and the second signal are below the predetermined threshold, the level is calculated from the average of the first and second signals.

[0040] In some embodiments, the sample is an in vitro sample. In some embodiments, the method includes determining the average of the first and second signals by determining the first and second levels corresponding to the first and second signals, and averaging the first and second levels. In some embodiments, the level of the hepatitis B virus is a concentration. In some embodiments, the level of the hepatitis B virus is a quantity. In some embodiments, one or both of the first and second amplicons contain a sequence from the S ORF of HBV. In some embodiments, one or both of the first and second amplicons contain a sequence from the P ORF of HBV. In some embodiments, one or both of the first and second amplicons contain a sequence from the overlapping portion of the S and P ORFs of HBV. In some embodiments, the first amplicon contains at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 consecutive nucleotides of one of SEQ ID NO: 2 or 3. In some embodiments, the first amplicon comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides of one of SEQ ID NOs: 20, 21, or 22. In some embodiments, the second amplicon comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides of SEQ ID NO: 41. In some embodiments, the second amplicon comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides of one of SEQ ID NOs: 34 or 35.

[0041] In some embodiments, the predetermined threshold is at a value where the expected random error is greater than or approximately equal to the expected error caused by the point mutation. In some embodiments, the predetermined threshold is at a value where the expected error caused by the point mutation is greater than or approximately equal to the expected random error. In some embodiments, the predetermined threshold corresponds to a concentration in the range of about 10IU / ml to about 200IU / ml. In some embodiments, the predetermined threshold is in the range of about 20IU / ml to about 40IU / ml, about 40IU / ml to about 60IU / ml, about 60IU / ml to about 80IU / ml, or about 80IU / ml to about 100IU / ml.

[0042] In some embodiments, the first label, the second label, or both are fluorescent. In some embodiments, at least one of the first and second labels is attached to a probe. In some embodiments, the probe comprises a quencher.

[0043] A kit or composition is also provided, comprising a probe oligomer disclosed herein. A kit or composition is also provided, comprising an amplification oligomer disclosed herein. In some embodiments, the kit or composition further comprises a probe oligomer disclosed herein.

[0044] In some embodiments, the kit or composition further comprises at least one, two, three, or four of a first amplification oligomer disclosed herein, a second amplification oligomer disclosed herein, a third amplification oligomer disclosed herein, or a fourth amplification oligomer disclosed herein.

[0045] In some embodiments, the kit or composition further comprises at least one, two, three, or four of a first capture oligomer disclosed herein, a second capture oligomer disclosed herein, a third capture oligomer disclosed herein, or a fourth capture oligomer disclosed herein.

[0046] In some embodiments, the kit or composition further comprises at least one or both of the fifth amplification oligomer disclosed herein, or the sixth amplification oligomer disclosed herein.

[0047] In some embodiments, the kit or composition further comprises an additional amplification oligomer disclosed herein, wherein if the kit or composition comprises a second amplification oligomer disclosed herein, the additional amplification oligomer is different from the second amplification oligomer.

[0048] In some embodiments, one, two or more oligomers disclosed herein are provided in a kit. In some embodiments, one, two or more oligomers disclosed herein are provided in a composition. In some embodiments, the composition is aqueous, frozen or lyophilized.

[0049] The section headings are provided for the convenience of the reader and do not limit the scope of the disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1A Alignment of exemplary target capture oligomers 0707b and 733 with HBV clone sequences representing genotypes AH is shown.

[0051] Figure 1B -C shows the alignment of exemplary target capture oligomers 1290 and 1168 with the 10 most common HBV database sequences at the corresponding positions, respectively. Rows are labeled with single letters (which do not necessarily correspond to the HBV genotype nomenclature) and indicate the frequency of matching sequences. Dots represent matches with oligomers, while mismatches are represented by letters. Figure 1B The Hth row and Figure 1C The series of N' in row G indicates an HBV sequence with an internal deletion.

[0052] FIG. 1D shows an alignment of exemplary oligomers with different HBV clone sequences in a region encompassing HBV positions 376-474. Figure 1D-2 is from Figure 1D-1 The continuation of the right end alignment.

[0053] Figure 2A An alignment of exemplary oligomers to different HBV clone sequences is shown, with differences shown relative to the 376-402T7 amplified oligomer (SEQ ID NO: 15).

[0054] FIG. 2B shows an alignment of an exemplary oligomer, a representative HBV genotype A sequence, and the HBV sequence of GenBank Accession No. AB674430, which contains a 108 nucleotide deletion.

[0055] FIG. 3 shows an alignment of exemplary oligomers with different HBV clone sequences in the region comprising HBV positions 640-720.

[0056] Figure 4A -D shows the emergence curves of the experiment in the absence (AB) and presence (CD) of the initial amplification oligos A376 and A35. Figure 4A and 4C Detection of the amplicon detected by the 408-435 probe oligomer is shown. Figure 4B and 4D Detection of amplicons detected by the 668A probe oligomer is shown. Arrows indicate traces for the 2,000 IU / ml sample and brackets indicate traces for samples with lower concentrations. The trace for the lowest concentration (2 IU / ml) is grey and the trace for the intermediate concentration (10 IU / ml) is black.

[0057] Figure 5 Shown are the observed log differences relative to expected quantitation for the exemplary quantitation method (white bars) compared to commercially available assays from Abbott (striped bars) and Roche (black bars) performed on the PEI genotype panel.

[0058] Fig. 6A A scatter plot is shown, demonstrating the reproducibility of HBV quantification.

[0059] Figure 6B -C show the calibration curves used to convert emergence times of amplicons 1 and 2, respectively, into log copies / ml. DETAILED DESCRIPTION

[0060] A. Definition

[0061] Before describing the present teachings in detail, it should be understood that the present disclosure is not limited to specific compositions or process steps, as these may vary. It should be noted that as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "an oligomer" includes a plurality of oligomers, etc.

[0062] It should be understood that there is an implied "about" before the temperature, concentration, time, etc. discussed in this disclosure, so that slight and insubstantial deviations are within the scope of the teachings herein. Generally, the term "about" represents an insubstantial change in the amount of a component of a composition, which has no significant effect on the activity or stability of the composition. Moreover, the use of "comprising", "containing", and "including" is not intended to be restrictive. It should be understood that the foregoing general description and detailed description are only exemplary and explanatory, and are not limitations on this teaching. To the extent that any material incorporated by reference is inconsistent with the stated content of the present disclosure, the stated content shall prevail.

[0063] Unless otherwise specified, embodiments in the specification described as “comprising” various components are also considered to be “consisting of” or “consisting essentially of” the components; embodiments in the specification described as “consisting of” the various components are also considered to be “comprising” or “consisting essentially of” the components; embodiments in the specification described as “consisting essentially of” the various components are also considered to be “consisting of” or “comprising” the components (this interchangeability does not apply to the use of these terms in the claims).

[0064] "Sample" includes any sample that may contain hepatitis B virus (HBV) or its components, such as nucleic acid or nucleic acid fragments. Samples include "biological samples", which include any tissue or material derived from living or dead people, which may contain HBV or target nucleic acids derived therefrom, including, for example, peripheral blood, plasma, serum, lymph nodes, gastrointestinal tissue (e.g., liver), or other body fluids or materials. Biological samples can be treated to physically or mechanically destroy tissue or cell structures, thereby releasing intracellular components into a solution, which can further contain enzymes, buffers, salts, detergents, etc., which are used to prepare biological samples for analysis using standard methods. Moreover, samples can include processed samples, such as those obtained by passing the sample over a filter device or passing the sample through a filter device, or after centrifugation, or by adhering to a medium, a matrix, or a support.

[0065] "Nucleic acid" refers to a polymeric compound comprising two or more covalently bonded nucleosides or nucleoside analogs with nitrogenous heterocyclic bases or base analogs, wherein the nucleosides are linked together by phosphodiester bonds or other bonds to form polynucleotides. Nucleic acids include RNA, DNA or chimeric DNA-RNA polymers or oligonucleotides and analogs thereof. Nucleic acid "backbone" can be composed of a variety of connections, including one or more of sugar-phosphodiester bonds, peptide-nucleic acid bonds (in "peptide nucleic acids" or PNAs, see, for example, International Patent Application Publication No. WO 95 / 32305), phosphorothioate bonds, methylphosphonate bonds or combinations thereof. The sugar moiety of nucleic acids can be ribose or deoxyribose, or similar compounds with known substitutions, such as 2'-methoxy substitutions and 2'-halide substitutions (e.g., 2'-F). The nitrogenous base may be a conventional base (A, G, C, T, U), an analog thereof (e.g., inosine, 5-methylisocytosine, isoguanine; see, e.g., The Biochemistry of the Nucleic Acids 5-36, Adams et al., ed., 11th ed., 1992; Abraham et al., 2007, BioTechniques 43:617-24), a derivative of a purine or pyrimidine base (e.g., N 4 -methyldeoxyguanosine, deaza- or aza-purine, deaza- or aza-pyrimidine, pyrimidine base with a substituent at position 5 or 6, purine base with an altered or replaced substituent at position 2, 6 and / or 8, such as 2-amino-6-methylaminopurine, 6 -methylguanine, 4-thio-pyrimidine, 4-amino-pyrimidine, 4-dimethylhydrazine-pyrimidine, and O 4-alkyl-pyrimidines, and pyrazolo-compounds, such as unsubstituted or 3-substituted pyrazolo[3,4-d]pyrimidines; U.S. Pat. Nos. 5,378,825, 6,949,367, and International Patent Application Publication No. WO 93 / 13121, each of which is incorporated herein by reference). Nucleic acids may include "abasic" residues, in which the backbone does not include one or more nitrogenous bases of the residues (see, e.g., U.S. Pat. No. 5,585,481, which is incorporated herein by reference). Nucleic acids may contain only conventional sugars, bases, and linkages found in RNA and DNA, or may include conventional components and substitutions (e.g., conventional bases linked by a 2'-methoxy backbone, or nucleic acids comprising a mixture of conventional bases and one or more base analogs). Nucleic acids may include "locked nucleic acids" (LNA), in which one or more nucleotide monomers have a bicyclic furanose unit locked in an RNA-mimicking sugar conformation that enhances hybridization affinity to complementary sequences in single-stranded RNA (ssRNA), single-stranded DNA (ssDNA), or double-stranded DNA (dsDNA) (Vester et al., Biochemistry 43: 13233-41, 2004, incorporated herein by reference). Nucleic acids may include modified bases to alter the function or behavior of the nucleic acid, such as the addition of a 3'-terminal dideoxynucleotide to prevent additional nucleotides from being added to the nucleic acid. Synthetic methods for preparing nucleic acids in vitro are well known in the art, although nucleic acids may be purified from natural sources using conventional techniques.

[0066] A sequence is a "hepatitis B virus sequence" if the sequence or its complement occurs at least about 90% or at least about 95% identical to any genotype, subtype or isolate of HBV, or contains no more than one mismatch, such that, for example, "14 consecutive nucleotides of a hepatitis B virus sequence" refers to a 14-mer that matches at least 13 of the 14 positions of a genotype, subtype or isolate of HBV or its complement. For purposes of determining whether a sequence conforms to a hepatitis B virus sequence, the presence of U is considered equivalent to T and vice versa. The target hybridization regions of the exemplary oligomers disclosed herein, the HBV-derived sequences of the in vitro transcripts disclosed herein, and subsequences thereof are also considered hepatitis B virus sequences. Thus, examples of hepatitis B virus sequences include SEQ ID NOs: 1-7, 20-28, 34-39, 49-64, 69-76, 82-84, and 96-125, as well as the complete HBV genome sequence referenced by the accession number in the sequence listing. In some embodiments, the genotype, subtype or isolate of HBV mentioned above is a known genotype, subtype or isolate of HBV, for example, which is present in a sequence database or publication available on the date of this disclosure. When referring to numerical positions in HBV nucleic acid, these positions refer to SEQ ID NO: 1, which is a HBV genotype A1 sequence. It should be understood that the positions of other genotypes may be different; for example, position 700 in SEQ ID NO: 1 is aligned with position 703 of the HBV genotype Fla sequence of SEQ ID NO: 101. The corresponding positions can be determined using an appropriate alignment algorithm, such as the Needleman-Wunsch algorithm with standard parameters.

[0067] When an oligomer comprises, for example, "at least 10 consecutive nucleotides of a specific SEQ ID NO" and "at least about 14 consecutive nucleotides of a hepatitis B virus sequence", the same nucleotides can be counted for both (i) and (ii), for example, the at least 14 consecutive nucleotides of the hepatitis B virus sequence can include any or all of the at least 10 consecutive nucleotides of the specified SEQ ID NO, to the extent consistent with the aforementioned definition of the hepatitis B virus sequence. Similarly, if every sequence of SEQ ID NO is present, then "the oligomer comprises a target hybridization sequence containing at least two" (or more) multiple specified SEQ ID NOs, whether or not they overlap. Thus, as a simplified example, CAT comprises both CA and AT.

[0068] For two molecules that "anneal to at least N common positions", it is meant that the molecules have hybridization sites that overlap with N or more nucleotides on the same or opposite strands of a target nucleic acid (e.g., HBV nucleic acid). For example, a first oligomer configured to specifically hybridize to positions 655-681 and a second oligomer configured to specifically hybridize to positions 679-699 anneal to three common positions (679, 680, and 681), regardless of whether (i) they are both configured to specifically hybridize to the same strand or (ii) one is configured to specifically hybridize to the sense or (+) strand and the other is configured to specifically hybridize to the antisense or (-) strand.

[0069] The term "polynucleotide" as used herein refers to a nucleic acid chain. Throughout the application, nucleic acids are designated from the 5'-end to the 3'-end. Synthetic nucleic acids, such as DNA, RNA, DNA / RNA chimeras (including when non-natural nucleotides or analogs are included therein), are typically synthesized "3'-to-5'", i.e., by adding nucleotides to the 5'-end of the growing nucleic acid.

[0070] As used herein, a "nucleotide" is a subunit of a nucleic acid consisting of a phosphate group, a 5-carbon sugar, and a nitrogenous base (also referred to herein as a "nucleobase"). The 5-carbon sugar found in RNA is ribose. In DNA, the 5-carbon sugar is 2'-deoxyribose. The term also includes analogs of these subunits, such as a methoxy group at the 2' position of ribose (also referred to herein as "2'-O-Me" or "2'-methoxy"). As used herein, a methoxy oligonucleotide containing a "T" residue has a methoxy group at the 2' position of the ribose moiety and a uracil at the base position of the nucleotide.

[0071] As used herein, "non-nucleotide units" are units that do not significantly participate in polymer hybridization. For example, these units do not participate in any significant hydrogen bonding with nucleotides, and exclude units having one of the five nucleotide bases or their analogs as components.

[0072] As used herein, "target nucleic acid" is a nucleic acid comprising a target sequence to be amplified. The target nucleic acid can be a DNA or RNA as described herein, and can be single-stranded or double-stranded. The target nucleic acid may include other sequences besides the target sequence, which may not be amplified.

[0073] As used herein, the term "target sequence" refers to a specific nucleotide sequence of a target nucleic acid to be amplified and / or detected. A "target sequence" includes a composite sequence that is complexed with an oligonucleotide (e.g., a priming oligonucleotide and / or a promoter oligonucleotide) during an amplification process (e.g., TMA). When the target nucleic acid is initially single-stranded, the term "target sequence" also refers to a sequence that is complementary to the "target sequence" present in the target nucleic acid. When the target nucleic acid is initially double-stranded, the term "target sequence" refers to both the sense (+) and antisense (-) strands. The (+) strand corresponds to a viral mRNA sequence, and (-) is its complement. The exemplary genotype A sequence of SEQ ID NO: 1 represents the (+) strand.

[0074] "Target hybridization sequence" is used herein to refer to the part of an oligomer configured to hybridize with a target nucleic acid sequence. In some embodiments, the target hybridization sequence is configured to specifically hybridize with a target nucleic acid sequence. The target hybridization sequence can be 100% complementary to the part of the target sequence to which they are configured to hybridize, but it is not necessary. The target hybridization sequence may also include inserted, deleted and / or substituted nucleotide residues relative to the target sequence. For example, when the target nucleic acid is a plurality of strains within a species, it may occur that the target hybridization sequence is less than 100% complementary to the target sequence, such as the case of an oligomer configured to hybridize with various genotypes of HBV. It should be understood that there are other reasons for configuring the target hybridization sequence to have less than 100% complementarity with the target nucleic acid.

[0075] As used herein, the term "target sequence" with respect to the HBV nucleic acid region refers to the process by which an oligonucleotide hybridizes with a target sequence in a manner that allows amplification and detection as described herein. In a preferred embodiment, the oligonucleotide is complementary to the targeted HBV nucleic acid sequence and does not contain mismatches. In another preferred embodiment, the oligonucleotide is complementary but contains 1, 2, 3, 4 or 5 mismatches with the targeted HBV nucleic acid sequence. In some embodiments, the oligonucleotide hybridized with the HBV nucleic acid sequence includes at least 10 to up to 50 nucleotides complementary to the target sequence. It should be understood that at least 10 and up to 50 are inclusive ranges, so as to include 10, 50 and every integer therebetween. In some embodiments, the oligomer specifically hybridizes with the target sequence.

[0076] The term "configured to" refers to the actual arrangement of the polynucleotide sequence configuration of the oligonucleotide target hybridization sequence of reference. For example, an amplification oligomer configured to produce a specific amplicon from a target sequence has a polynucleotide sequence that hybridizes with the target sequence and can be used for an amplification reaction to produce an amplicon. Also as an example, an oligonucleotide configured to specifically hybridize with a target sequence has a polynucleotide sequence that specifically hybridizes with a reference sequence under stringent hybridization conditions.

[0077] As used herein, the term "configured to specifically hybridize with..." refers to a polynucleotide sequence in which the target hybridization region of an amplification oligonucleotide, a detection probe, or other oligonucleotide is designed to have a sequence of a HBV target region that can be targeted for reference. Such oligonucleotides are not limited to targeting only this sequence, but are very useful as a composition in a kit or in a method for targeting HBV target nucleic acids. Oligonucleotides are designed to be used as components of an assay for amplifying and detecting HBV from a sample, and are therefore designed to target HBV in the presence of other nucleic acids commonly found in a test sample. "Specifically hybridizing with..." does not mean complete hybridization, because some small levels of hybridization with non-target nucleic acids may occur, as understood in the art. On the contrary, "specifically hybridizing with..." refers to an oligonucleotide that is configured to function in an assay to primarily hybridize with a target, so that accurate detection of a target nucleic acid in a sample can be determined. "Upstream" refers to a position closer to the 5' end of a (+) chain (or the 3' end of a (-) chain) than a given position. "Downstream" refers to a position closer to the 3' end of a (+) chain (or the 5' end of a (-) chain) than a given position.

[0078] As used herein, the term "fragment" with respect to a targeted HBV nucleic acid refers to a piece of continuous nucleic acid. In certain embodiments, the fragment includes continuous nucleotides from the HBV RNA corresponding to SEQ ID NO: 1, wherein the number of continuous nucleotides in the fragment is less than the number of the complete sequence corresponding to SEQ ID NO: 1.

[0079] As used herein, the term "region" refers to a portion of a nucleic acid, wherein the portion is smaller than the entire nucleic acid. For example, when the nucleic acid in the reference is an oligonucleotide promoter primer, the term "region" can be used to refer to a smaller promoter portion of the entire oligonucleotide. Similarly, and by way of example only, when the nucleic acid is an HBV nucleic acid, the term "region" can be used to refer to a smaller region of the nucleic acid, wherein the smaller region is targeted by one or more oligonucleotides of the present disclosure. As another non-limiting example, when the nucleic acid in the reference is an amplicon, the term region can be used to refer to a smaller nucleotide sequence for hybridization identified by the target hybridization sequence of the probe.

[0080] The interchangeable terms "oligomer", "oligo" and "oligonucleotide" refer to nucleic acids having generally less than 1,000 nucleotide (nt) residues, including polymers having a lower limit of about 5nt residues and an upper limit of about 500 to 900nt residues. In some embodiments, the size range of oligonucleotides has a lower limit of about 12 to 15nt and an upper limit of about 50 to 600nt, and the range of other embodiments has a lower limit of about 15 to 20nt and an upper limit of about 22 to 100nt. Oligonucleotides can be purified from naturally occurring sources, or can be synthesized using any of a variety of well-known enzymes or chemical methods. The term oligonucleotide does not represent any specific function of an agent; on the contrary, it is generally used to cover all such agents described herein. Oligonucleotides can be used for a variety of different functions. For example, if it is specific for and capable of hybridizing to a complementary strand and can be further extended in the presence of a nucleic acid polymerase, it can function as a primer; if it contains a sequence recognized by an RNA polymerase and allows transcription (e.g., a T7 primer), it can function as a primer and provide a promoter; and if it is capable of hybridizing to a target nucleic acid or an amplicon thereof, it can function to detect the target nucleic acid and further provide a detectable portion (e.g., a fluorophore).

[0081] As used herein, an oligonucleotide that "substantially corresponds to" a specific reference nucleic acid sequence means that the oligonucleotide is sufficiently similar to the reference nucleic acid sequence so that the oligonucleotide has hybridization properties similar to the reference nucleic acid sequence because it will hybridize with the same target nucleic acid sequence under stringent hybridization conditions. It will be understood by those skilled in the art that a "substantially corresponding oligonucleotide" can be different from the reference sequence and still hybridize with the same target nucleic acid sequence. It should also be understood that the first nucleic acid corresponding to the second nucleic acid includes its RNA or DNA equivalent and its DNA / RNA chimera, and includes its complement, unless the context clearly indicates otherwise. This change in nucleic acid can be expressed in terms of the percentage of identical bases within the sequence or the percentage of fully complementary bases between the probe or primer and its target sequence. Therefore, in certain embodiments, if these percentages of base identity or complementarity are 100% to about 80%, the oligonucleotide "substantially corresponds to" the reference nucleic acid sequence. In some embodiments, the percentage is 100% to about 85%. In some embodiments, the percentage is 100% to about 90%, for example, 100% to about 95%. Similarly, the region of a nucleic acid or amplified nucleic acid may be referred to herein as corresponding to a reference nucleic acid sequence. Those skilled in the art will appreciate that various modifications of the hybridization conditions may be required for various percentages of complementarity to allow hybridization to a specific target sequence without causing unacceptable levels of non-specific hybridization.

[0082] As used herein, "blocking moiety" is a material for "blocking" the 3'-end of an oligonucleotide or other nucleic acid so that it cannot be effectively extended by a nucleic acid polymerase. An oligomer that is not intended to be extended by a nucleic acid polymerase may include a blocking group that replaces 3'OH to prevent oligomer enzyme-mediated extension in an amplification reaction. For example, an amplification oligomer and / or a detection probe that blocks during amplification may not have a functional 3'OH, but may include one or more blocking groups located at or near the 3' end. In some embodiments, the blocking group near the 3' end may be within 5 residues at the 3' end and be large enough to limit the combination of a polymerase with an oligomer. In other embodiments, the blocking group is covalently linked to the 3' end. Many different chemical groups may be used to block the 3' end, such as alkyl, non-nucleotide linker, alkanediol dideoxynucleotide residues, and cordycepin.

[0083] "Amplification oligomer" is an oligomer whose at least 3'-end is complementary to the target nucleic acid, and hybridizes with the target nucleic acid or its complement, and participates in the nucleic acid amplification reaction. The example of an amplification oligomer is a "primer", which hybridizes with the target nucleic acid and contains a 3'OH end extended by a polymerase during the amplification process. In some embodiments, the 5' region of the amplification oligonucleotide may include a promoter sequence that is not complementary to the target nucleic acid (which may be referred to as a "promoter primer"). Another example of an amplification oligomer is an oligomer that is not extended by a polymerase (for example, because it has a 3' blocking end) but participates in or promotes amplification. For example, the 5' region of the amplification oligonucleotide may include a promoter sequence that is not complementary to the target nucleic acid (which may be referred to as a "promoter provider"). It will be understood by those skilled in the art that the amplification oligomer that acts as a primer can be modified to include a 5' promoter sequence, thereby acting as a promoter primer. The end of the incorporation of the 3' blocking further modifies the promoter primer, which is now able to hybridize with the target nucleic acid and provide an upstream promoter sequence for initiating transcription, but does not provide a primer for oligonucleotide extension. The oligonucleotide of this modification is referred to as " promoter provider " oligomer in this article.The size range of amplification oligonucleotide includes those of about 10 to about 70nt length (excluding any promoter sequence or poly-A tail) and contains at least 10 continuous bases, or even at least 12 continuous bases complementary to the target nucleic acid sequence region (or its complementary strand).The target sequence that continuous bases are combined with amplification oligomer is at least 80%, or at least 90%, or fully complementary.Amplification oligomer can optionally include modified nucleotides or analogs, or participate in amplification reaction but not complementary to target nucleic acid or template sequence or other nucleotides included.It should be understood that when referring to the length range of oligonucleotide, amplicon or other nucleic acid, the range includes all integers (for example, a length of 19-25 continuous nucleotides includes 19,20,21,22,23,24 and 25).

[0084] As used herein, a "promoter" is a specific nucleic acid sequence that is recognized by a DNA-dependent RNA polymerase ("transcriptase") as a signal to bind to nucleic acid and initiate RNA transcription at a specific site.

[0085] As used herein, "promoter provider" or "provider" refers to an oligonucleotide comprising a first and a second region, and it is modified to prevent the initiation of DNA synthesis from its 3'-end. The "first region" of the promoter provider oligonucleotide comprises a base sequence that hybridizes with a DNA template, wherein the hybridization sequence is located at the 3' of the promoter region, but is not necessarily adjacent to the promoter region. The hybridization portion of the promoter oligonucleotide is usually at least 10 nucleotides in length and can be extended to 50 or more nucleotides in length. The "second region" comprises a promoter sequence for RNA polymerase. The promoter oligonucleotide is designed so that it cannot be extended by RNA or DNA-dependent DNA polymerases (e.g., reverse transcriptase). In some embodiments, a blocking portion is included at its 3'-end as described above. As described herein, a "T7 provider" is a blocked promoter provider oligonucleotide that provides an oligonucleotide sequence recognized by a T7 RNA polymerase.

[0086] "Terminator oligonucleotide" is an oligonucleotide comprising a base sequence that is substantially complementary to a sequence in a target nucleic acid near the 5'-end of the target region, to "terminate" primer extension of a nascent nucleic acid, which includes an initiating oligonucleotide, thereby providing a definite 3'-end of a nascent nucleic acid chain. The terminator oligonucleotide is designed to hybridize with the target nucleic acid at a position sufficient to achieve the desired 3'-end of the nascent nucleic acid chain. Depending on its design, the positioning of the terminator oligonucleotide is flexible. The terminator oligonucleotide can be modified or unmodified. In certain embodiments, the terminator oligonucleotide is synthesized using at least one or more 2'-O-ME ribonucleotides. These modified nucleotides have demonstrated higher thermal stability of complementary duplexes. 2'-O-ME ribonucleotides also serve to increase the exonuclease resistance of the oligonucleotide, thereby increasing the half-life of the modified oligonucleotide. (See, e.g., Majlessi et al., Nucleic Acids Res. 26: 2224-9, 1988, incorporated herein by reference.) In addition to or in place of 2'-O-Me ribonucleotides, other modifications as described elsewhere herein may be used. For example, the terminator oligonucleotide may comprise a PNA or LNA. (See, e.g., Petersen et al., J. Mol. Recognit. 13: 44-53, 2000, incorporated herein by reference.) The terminator oligonucleotides of the present disclosure typically include a blocking portion at their 3'-end to prevent extension. The terminator oligonucleotide may also include a protein or peptide attached to the oligonucleotide to terminate further extension of the nascent nucleic acid chain by the polymerase. The terminator oligonucleotides of the present disclosure are typically at least 10 bases in length, and may extend up to 15, 20, 25, 30, 35, 40, 50 or more nucleotides in length. Although a terminator oligonucleotide will typically or necessarily include a 3'-blocking moiety, a "3'-blocked" oligonucleotide is not necessarily a terminator oligonucleotide.

[0087] "Amplification" refers to any known procedure for obtaining multiple copies of a target nucleic acid sequence or its complement or its fragment. Multiple copies can be referred to as amplicons or amplification products. The amplification of a "fragment" refers to the generation of an amplified nucleic acid containing less than a complete target nucleic acid or its complement, such as produced by using an amplification oligonucleotide, which hybridizes and polymerizes from an internal position of the target nucleic acid. Known amplification methods include, for example, replicase-mediated amplification, polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA) and transcription-mediated or transcription-related amplification. Replicase-mediated amplification uses self-replicating RNA molecules and replicase such as QB-replicase (see, for example, U.S. Patent No. 4,786,600, incorporated herein by reference). PCR amplification uses DNA polymerase, primer pairs and thermal cycles to synthesize multiple copies of two complementary chains of dsDNA or from cDNA (see, for example, U.S. Patent Nos. 4,683,195; 4,683,202; and 4,800,159; each incorporated herein by reference). LCR amplification uses four or more different oligonucleotides to amplify the target and its complementary strand by using multiple cycles of hybridization, ligation, and denaturation (see, e.g., U.S. Pat. Nos. 5,427,930 and 5,516,663, each of which is incorporated herein by reference). SDA uses primers containing a restriction endonuclease recognition site and an endonuclease that cleaves one strand of a hemi-modified DNA duplex including the target sequence, whereby amplification occurs in a series of primer extension and strand displacement steps (see, e.g., U.S. Pat. Nos. 5,422,252; 5,547,861; and 5,648,211; each of which is incorporated herein by reference).

[0088] As used herein, the term "linear amplification" refers to an amplification mechanism that is designed to produce an increase in a target nucleic acid that is linearly proportional to the amount of the target nucleic acid in the reaction. For example, a transcription-related reaction can be used to prepare multiple RNA copies from a DNA target, where the increase in the number of copies can be described by a linear factor (e.g., the starting copy of the template × 100). In some embodiments, the first-stage linear amplification in a multi-stage amplification program increases the starting number of the target nucleic acid chain or its complement by at least 10 times, such as at least 100 times, or 10 to 1,000 times before the start of the second-stage amplification reaction. An example of a linear amplification system is "linear amplification of DNA based on T7" (TLAD; see Liu et al., BMC Genomics, 4: Art. May 9, 2003 No. 19). Other methods are known, for example, from U.S. Pat. No. 9,139,870, or disclosed herein. Therefore, the term "linear amplification" refers to an amplification reaction that does not result in an exponential amplification of a target nucleic acid sequence. The term "linear amplification" does not refer to methods that simply make a single copy of a nucleic acid strand, such as the transcription of an RNA molecule into a single cDNA molecule, as in the case of reverse transcription (RT)-PCR.

[0089] As used herein, the term "exponential amplification" refers to a nucleic acid amplification designed to produce an increase in target nucleic acid, geometrically proportional to the amount of target nucleic acid in the reaction. For example, PCR produces a DNA strand for each original target strand and each synthetic strand present. Similarly, transcription-related amplification produces multiple RNA transcripts for each original target strand and each subsequently synthesized strand. Amplification is exponential because the synthesized strands are used as templates in subsequent amplification rounds. An amplification reaction does not actually need to produce an exponentially increasing amount of nucleic acid to be considered an exponential amplification, as long as the amplification reaction is designed to produce such an increase.

[0090] "Transcription-associated amplification" or "transcription-mediated amplification" (TMA) refers to nucleic acid amplification that uses an RNA polymerase to produce multiple RNA transcripts from a nucleic acid template. These methods typically use RNA polymerase, DNA polymerase, deoxyribonucleoside triphosphates, ribonucleoside triphosphates, and template-complementary oligonucleotides, which include a promoter sequence, such as a T7 promoter, and optionally may include one or more other oligonucleotides. When an oligomer containing a T7 promoter is used, it may be referred to as a "T7 primer" or "T7 oligomer"; other primers / oligomers may be referred to as "non-T7" or "NT7" primers / oligomers. The TMA method and the single primer transcription-associated amplification method are embodiments of amplification methods for detecting HBV target sequences as described herein. Variations of transcription-related amplification are well known in the art, as previously disclosed in detail (see, e.g., U.S. Pat. Nos. 4,868,105; 5,124,246; 5,130,238; 5,399,491; 5,437,990; 5,554,516; and 7,374,885; and International Patent Application Publication Nos. WO 88 / 01302; WO 88 / 10315; and WO 95 / 03430; each incorporated herein by reference). One of ordinary skill in the art will appreciate that the disclosed compositions can be used in amplification methods based on polymerase extension of oligomer sequences.

[0091] As used herein, the term "real-time TMA" refers to single-primer transcription-mediated amplification ("TMA") of a target nucleic acid monitored by real-time detection.

[0092] As used herein, the term "amplicon" or "amplification product" refers to a nucleic acid molecule produced during an amplification procedure that is complementary or homologous to a sequence contained within a target sequence. The complementary or homologous sequence of an amplicon is sometimes referred to herein as a "target-specific sequence". The amplicon produced using the amplification oligomers disclosed herein may contain non-target-specific sequences. The amplicon may be double-stranded or single-stranded, and may include DNA, RNA, or both. For example, a DNA-dependent RNA polymerase transcribes single-stranded amplicons from double-stranded DNA in a transcription-mediated amplification procedure. These single-stranded amplicons are RNA amplicons, and may be either strand of a double-stranded complex, depending on how the amplification oligomer is configured. Therefore, the amplicon may be a single-stranded RNA. RNA-dependent DNA polymerases synthesize DNA strands complementary to an RNA template. Therefore, the amplicon may be a double-stranded DNA and RNA hybrid. RNA-dependent DNA polymerases typically include ribonuclease activity, or are used in combination with ribonucleases, which degrade RNA strands. Therefore, the amplicon may be a single-stranded DNA. RNA-dependent DNA polymerases and DNA-dependent DNA polymerases synthesize complementary DNA strands from DNA templates. Therefore, the amplicon can be a double-stranded DNA. RNA-dependent RNA polymerase synthesizes RNA from an RNA template. Therefore, the amplicon can be a double-stranded RNA. DNA-dependent RNA polymerase synthesizes RNA from a double-stranded DNA template, also known as transcription. Therefore, the amplicon can be a single-stranded RNA. Amplicons and methods of producing amplicons are known to those skilled in the art. For convenience herein, single-stranded RNA or single-stranded DNA may represent the amplicon produced by the amplification oligomer combination of the present disclosure. This representation is not meant to limit the amplicon to the representation shown. A technician in possession of the present disclosure will use amplification oligomers and polymerases to produce any of a variety of types of amplicons, all of which are within the spirit and scope of the present disclosure.

[0093] As used herein, "non-target specific sequence" refers to a region of an oligomer sequence, wherein the region is not stably hybridized with a target sequence under standard hybridization conditions. Oligomers with non-target specific sequences include, but are not limited to, promoter primers and molecular beacons. Amplification oligomers may contain sequences that are not complementary to a target or template sequence; for example, the 5' region of a primer may include a promoter sequence (referred to as a "promoter primer") that is not complementary to a target nucleic acid. It will be appreciated by those skilled in the art that an amplification oligomer that acts as a primer may be modified to include a 5' promoter sequence, thereby acting as a promoter primer. Similarly, a promoter primer may be modified by removing a promoter sequence or synthesizing in the absence of a promoter sequence, and still acts as a primer. A 3' blocked amplification oligomer may provide a promoter sequence and serve as a template for polymerization (referred to as a "promoter provider"). Therefore, an amplicon produced by an amplification oligomer member such as a promoter primer will contain a target-specific sequence and a non-target-specific sequence.

[0094] "Detection probe," "detection oligonucleotide," "probe oligomer," and "detection probe oligomer" are used interchangeably and refer to a nucleic acid oligomer that specifically hybridizes to a target sequence in a nucleic acid or an amplified nucleic acid under conditions that promote hybridization to allow detection of the target sequence or the amplified nucleic acid. Detection can be direct (e.g., the probe hybridizes directly to its target sequence) or indirect (e.g., the probe is attached to its target through an intermediate molecular structure). The detection probe can be DNA, RNA, an analog thereof, or a combination thereof (e.g., a DNA / RNA chimera), which can be labeled or unlabeled. The detection probe can further include alternative backbone connections, such as, for example, 2'-O-methyl connections. The "target sequence" of a detection probe generally refers to a smaller nucleic acid sequence region within a larger nucleic acid sequence that specifically hybridizes to at least a portion of the probe oligomer through standard base pairing. Detection probes may comprise target-specific sequences and other sequences that contribute to the three-dimensional conformation of the probe (see, e.g., U.S. Patent Nos. 5,118,801; 5,312,728; 6,849,412; 6,835,542; 6,534,274; and 6,361,945; and U.S. Patent Application Publication No. 20060068417; each of which is incorporated herein by reference).

[0095] By "stable" or "stable for detection" is meant that the temperature of the reaction mixture is at least 2°C below the melting temperature of the nucleic acid duplex.

[0096] As used herein, "label" refers to a part or compound directly or indirectly connected to a probe that is detected or causes a detectable signal. Direct labeling can occur by a key or interaction connecting the label to the probe, including covalent bonds or non-covalent interactions, such as hydrogen bonds, hydrophobic and ionic interactions, or forming chelates or coordination complexes. Indirect labeling can occur by using a bridging part or "linker", such as a binding pair member, an antibody or another oligomer, which is directly or indirectly labeled and can amplify a detectable signal. Labeling includes any detectable part, such as a radionuclide, a ligand (e.g., biotin, avidin), an enzyme or enzyme substrate, a reactive group or a chromophore (e.g., a dye, particle or bead that imparts a detectable color), a luminescent compound (e.g., bioluminescent, phosphorescent or chemiluminescent label) or a fluorophore. Labeling can be detected in a homogeneous assay, wherein the labeled probe combined in the mixture exhibits a detectable change different from the uncombined labeled probe, such as instability or differential degradation properties. "Uniform detectable labels" can be detected without physically removing the bound from the unbound form of the label or labeled probe (see, e.g., U.S. Pat. Nos. 5,283,174; 5,656,207; and 5,658,737; each of which is incorporated herein by reference). Labels include chemiluminescent compounds, such as acridinium ester ("AE") compounds including standard acridinium esters and derivatives (see, e.g., U.S. Pat. Nos. 5,656,207; 5,658,737; and 5,639,604; each of which is incorporated herein by reference). Methods and syntheses for attaching labels to nucleic acids and detecting labels are well known. (See, e.g., Sambrook et al., Molecular Cloning. A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), Chapter 10, incorporated herein by reference. See also U.S. Pat. Nos. 5,658,737; 5,656,207; 5,547,842; 5,283,174; and 4,581,333; each incorporated herein by reference.) More than one label and more than one type of label may be present on a particular probe, or detection may use a mixture of probes, each labeled with a compound that produces a detectable signal (see, e.g., U.S. Pat. Nos. 6,180,340 and 6,350,579, each incorporated herein by reference).

[0097] "Capture probe," "capture oligonucleotide," "capture oligomer," "target capture oligomer," and "capture probe oligomer" are used interchangeably and refer to a nucleic acid oligomer that specifically hybridizes to a target sequence in a target nucleic acid through standard base pairing and attaches to a binding partner on an immobilized probe to capture the target nucleic acid to a support. One example of a capture oligomer includes two binding regions: a sequence binding region (e.g., a target-specific portion) and an immobilized probe binding region, typically on the same oligomer, although the two regions can be present on two different oligomers bound together by one or more linkers. Another embodiment of a capture oligomer uses a target sequence binding region that includes a random or non-random poly-GU, poly-GT, or poly-U sequence to non-specifically bind to a target nucleic acid and attach it to an immobilized probe on a support.

[0098] As used herein, "immobilized oligonucleotide", "immobilized probe", "immobilized binding partner", "immobilized oligomer" or "immobilized nucleic acid" refers to a nucleic acid binding partner that directly or indirectly connects a capture oligomer to a support. The immobilized probe connected to the support facilitates the separation of the target bound by the capture probe from unbound materials in the sample. One embodiment of the immobilized probe is an oligomer connected to a support, which facilitates the separation of the bound target sequence from unbound materials in the sample. The support can include known materials, such as a matrix and particles free in solution, which can be made of nitrocellulose, nylon, glass, polyacrylate, mixed polymers, polystyrene, silane, polypropylene, metal or other compositions, one embodiment of which is a magnetically attractable particle. The support can be a monodisperse magnetic sphere (e.g., uniform size + 5%), to which the immobilized probe is directly connected (via covalent linkage, chelation or ionic interaction), or indirectly connected (via one or more linkers), wherein the connection or interaction between the probe and the support is stable during hybridization conditions.

[0099] "Complementary" refers to the nucleotide sequence of similar regions of two single-stranded nucleic acids, or two different regions of the same single-stranded nucleic acid, with a nucleotide base composition that allows the single-stranded regions to hybridize together in a stable double-stranded hydrogen bonding region under stringent hybridization or amplification conditions. Through standard nucleic acid base pairing (e.g., G:C, A:T or A:U pairing), the sequences that hybridize to each other can be completely complementary or partially complementary to the expected target sequence. "Sufficiently complementary" refers to a continuous sequence that can hybridize with another sequence through hydrogen bonding between a series of complementary bases, and the complementary bases can be complementary at every position in the sequence through standard base pairing, or can contain one or more residues, including non-complementary abasic residues. Sufficiently complementary continuous sequences are usually at least 80%, or at least 90% complementary to the sequence that the oligomer intends to specifically hybridize. "Sufficiently complementary" sequences allow nucleic acid oligomers to stably hybridize with their target sequences under appropriate hybridization conditions, even if the sequences are not completely complementary. Nucleotide sequences are "fully" complementary when a contiguous sequence of nucleotides in one single-stranded region is capable of pairing with a similar sequence of nucleotides in another single-stranded region to form a series of "canonical" or "Watson-Crick" hydrogen-bonded base pairs such that A pairs with U or T, and C pairs with G (see, e.g., Sambrook et al., Molecular Cloning. A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), §§1.90-1.91, 7.37-7.57, 9.47-9.51 and 11.47-11.57, in particular §§9.50-9.51, 11.12-11.13, 11.45-11.47 and 11.55-11.57, incorporated herein by reference). It should be understood that ranges of percent identity include all integers and fractions (eg, at least 90% includes 90, 91, 93.5, 97.687, etc.) Reference to the "complement" of a particular sequence is generally intended to refer to fully complementary sequences unless the context dictates otherwise.

[0100] A "wobble" base pair is when a G pairs with either a U or a T.

[0101] "Preferential hybridization" or "specific hybridization" means that under stringent hybridization assay conditions, a probe hybridizes to its target sequence or copies thereof to form stable probe:target hybrids while minimizing the formation of stable probe:non-target hybrids. Thus, the probe hybridizes to the target sequence or copies thereof to a sufficiently greater extent than to non-target sequences to enable one of ordinary skill in the art to accurately detect or quantify RNA copies or complementary DNA (cDNA) of the target sequence formed during amplification. Appropriate hybridization conditions are well known in the art and can be predicted based on sequence composition or can be determined by use of routine testing procedures (see, e.g., Sambrook et al., Molecular Cloning. A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), §§1.90-1.91, 7.37-7.57, 9.47-9.51 and 11.47-11.57, in particular §§9.50-9.51, 11.12-11.13, 11.45-11.47 and 11.55-11.57, incorporated herein by reference).

[0102] "Nucleic acid hybrid", "hybrid" or "duplex" refers to a nucleic acid structure containing a double-stranded hydrogen bonding region, wherein each strand is complementary to the other strand, and wherein the region is sufficiently stable under stringent hybridization conditions to be detected by means including, but not limited to, chemiluminescent or fluorescent detection, autoradiography, or gel electrophoresis. Such hybrids may comprise RNA: RNA, RNA: DNA, or DNA: DNA duplex molecules.

[0103] "Sample preparation" refers to any step or method for treating a sample for subsequent amplification and / or detection of HBV nucleic acids present in the sample. A sample can be a complex mixture of components, wherein the target nucleic acid is a minority component. Sample preparation may include any known method for concentrating components (such as microorganisms or nucleic acids) from a larger sample volume, such as by filtering airborne or waterborne particles from a larger volume of sample, or by using standard microbiological methods to separate microorganisms from a sample. Sample preparation may include physical destruction and / or chemical lysis of cell components to release intracellular components into a substantially aqueous or organic phase and remove debris, such as by using filtration, centrifugation or adsorption. Sample preparation may include the use of nucleic acid oligonucleotides, which selectively or non-specifically capture target nucleic acids and separate them from other sample components (e.g., as described in U.S. Patent No. 6,110,678 and International Patent Application Publication No. WO 2008 / 016988, each incorporated herein by reference).

[0104] "Isolation" or "purification" refers to the removal or separation of one or more components of a sample from other sample components. Sample components typically include target nucleic acids, typically in an aqueous phase, which may also include cell fragments, proteins, carbohydrates, lipids, and other nucleic acids. "Isolation" or "purification" does not imply any degree of purification. Typically, separation or purification removes at least 70%, or at least 80%, or at least 95% of the target nucleic acid from other sample components.

[0105] As used herein, "DNA-dependent DNA polymerase" is an enzyme that synthesizes a complementary DNA copy from a DNA template. Examples are DNA polymerase I from Escherichia coli, bacteriophage T7 DNA polymerase, or DNA polymerases from bacteriophages T4, Phi-29, M2, or T5. DNA-dependent DNA polymerases can be naturally occurring enzymes isolated or recombinantly expressed from bacteria or bacteriophages, or can be modified or "evolved" forms that have been engineered to have certain desired characteristics, such as thermal stability, or the ability to recognize or synthesize DNA chains from various modified templates. All known DNA-dependent DNA polymerases require complementary primers to initiate synthesis. It is known that under suitable conditions, DNA-dependent DNA polymerases can synthesize complementary DNA copies from RNA templates. RNA-dependent DNA polymerases also typically have DNA-dependent DNA polymerase activity.

[0106] As used herein, "DNA-dependent RNA polymerase" or "transcriptase" is an enzyme that synthesizes multiple RNA copies from a double-stranded or partially double-stranded DNA molecule having a promoter sequence that is usually double-stranded. RNA molecules ("transcripts") are synthesized in the 5'- to 3'-direction, starting from a specific position just downstream of the promoter. Examples of transcriptases are DNA-dependent RNA polymerases from Escherichia coli and bacteriophages T7, T3, and SP6.

[0107] As used herein, an "RNA-dependent DNA polymerase" or "reverse transcriptase" ("RT") is an enzyme that synthesizes a complementary DNA copy from an RNA template. All known reverse transcriptases also have the ability to make a complementary DNA copy from a DNA template; therefore, they are both RNA- and DNA-dependent DNA polymerases. RT may also have RNase H activity. Reverse transcriptases require primers to initiate synthesis using both RNA and DNA templates.

[0108] By "thermophilic" is meant an enzyme (e.g., a polymerase) that exhibits optimal activity at temperatures greater than about 45°C, such as at temperatures in the range of about 50°C to 99°C. In some embodiments, the thermophilic enzyme does not lose more than 50% of its activity after incubation at 60°C for 20 minutes. In some embodiments, the thermophilic enzyme is obtained or derived from a thermophilic organism, such as an organism having an optimal growth temperature greater than or equal to about 45°C, such as greater than or equal to about 50°C.

[0109] As used herein, a "selective ribonuclease" is an enzyme that degrades the RNA portion of an RNA:DNA duplex, rather than single-stranded RNA, double-stranded RNA, or DNA. An exemplary selective ribonuclease is ribonuclease H. Enzymes with the same or similar activity as ribonuclease H may also be used. The selective ribonuclease may be an endonuclease or an exonuclease. Most reverse transcriptases contain ribonuclease H activity in addition to polymerase activity. However, other sources of ribonuclease H are available without the associated polymerase activity. Degradation may result in separation of the RNA from the RNA:DNA complex. Alternatively, the selective ribonuclease may simply cut the RNA at a different location, causing a portion of the RNA to melt or allowing the enzyme to unwind portions of the RNA. Other enzymes that selectively degrade the RNA target sequence or RNA product of the present disclosure are readily apparent to those of ordinary skill in the art.

[0110] As used herein, a "standard curve" is a representation of some time-dependent markers relating to (1) the pre-amplified amount of a polynucleotide, and (2) the amplified amount of the corresponding amplicon. For example, a standard curve can be a graph with a known number of input template molecules plotted on the x-axis, and the time values ​​required for the amplification reaction to reach a certain level of detectable amplicon production plotted on the y-axis. A standard curve is typically generated using a control polynucleotide standard containing a known number of polynucleotide templates. The standard curve can be stored in electronic form or can be represented graphically. The pre-amplified amount of the analyte polynucleotide in the test sample can be determined by comparing the measured time-dependent values ​​obtained for the test sample with the standard curve, which is familiar to those of ordinary skill in the art.

[0111] In the context of amplification and / or detection systems, the term "specificity" is used herein to refer to a characteristic of a system that describes its ability to distinguish between target and non-target sequences, depending on the sequence and assay conditions. In the context of nucleic acid amplification, specificity generally refers to the ratio of the number of specific amplicons produced to the number of byproducts (e.g., signal-to-noise ratio). In the context of detection, specificity generally refers to the ratio of the signal generated from the target nucleic acid to the signal generated by the non-target nucleic acid.

[0112] The term "sensitivity" is used herein to refer to the accuracy with which a nucleic acid amplification reaction can be detected or quantified. The sensitivity of an amplification reaction is generally a measure of the minimum number of copies of a target nucleic acid that can be reliably detected in an amplification system, and will depend, for example, on the specificity of the detection assay and amplification reaction employed, e.g., the ratio of a specific amplicon to byproducts.

[0113] As used herein, the terms "relative light unit" ("RLU") and "relative fluorescence unit" ("RFU") refer to arbitrary units of measurement that represent the relative number of photons emitted by a sample at a given wavelength or wavelength band. The measured value of an RLU or RFU varies with the characteristics of the detector used for the measurement.

[0114] As used herein, the terms "T time", "emergence time" and "emergence time" are interchangeable and represent the threshold time or emergence time of a signal in a real-time graph of assay data. The T time value estimates the time at which amplicon generates a specific threshold value through which it passes in a real-time amplification reaction. The T time and the algorithm for calculating and using the T time value are described in Light et al., U.S. Patent Publication No. 2006 / 0276972, paragraphs

[0517] to

[0538] , the disclosure of which is incorporated herein by reference. A curve fitting program is applied to normalized and background adjusted data. Curve fitting is performed only for a portion of data between a predetermined lower limit and an upper limit. After finding the curve of the fitted data, the goal is to estimate the time corresponding to the point at which the curve or its projection intersects with a predetermined threshold. In one embodiment, the threshold value of the normalized data is 0.11. The upper and lower limits are determined empirically, wherein the range of the curve fit to various control data sets exhibits the minimum variability in the time associated with a given threshold. For example, in one embodiment, the lower limit is 0.04 and the upper limit is 0.36. The curve is applicable to data extending from the first data point below the lower limit to the first data point exceeding the upper limit. Next, determine whether the slope of the fit is statistically significant. For example, if the p-value for the first-order coefficient is less than 0.05, the fit is considered significant and processing continues. If not, processing stops. Alternatively, the validity of the data can be determined by R 2 The slope m and intercept b of the linear curve y=mx+b of the fitted curve are determined. With this information, T time can be determined as follows: T time=(threshold-b) / m.

[0115] Unless otherwise stated, a concentration of 1 IU / ml HBV corresponds to 5 copies / ml HBV nucleic acid. 1 log copy / ml HBV nucleic acid corresponds to 10 copies / ml, 2 log copies / ml HBV nucleic acid corresponds to 100 copies / ml, and so on, i.e. n log copies / ml equals 10 n copies / ml.

[0116] Unless otherwise noted, oligo sequences appearing in the following table follow the following convention: lower case letters indicate 2'-O-methyl RNA, upper case letters indicate DNA. "(c9)" indicates a -(CH2)9-linker. Unless otherwise noted, in vitro transcript (IVT) sequences are RNA.

[0117] References, particularly in the claims, to "the sequence of SEQ ID NO: X" refer to the base sequence of the corresponding sequence listing entry, and do not require backbone identity (e.g., RNA, 2'-O-Me RNA, or DNA) unless otherwise indicated. In addition, unless otherwise indicated, T and U residues are considered interchangeable for the purposes of sequence listing entries, e.g., a sequence can be considered identical to SEQ ID NO: 2 regardless of whether the residue at position 6 is T or U.

[0118] B. Oligomers, Compositions, and Kits

[0119] The present disclosure provides oligomers, compositions, and kits that can be used to amplify, detect, or quantify HBV from a sample.

[0120] In some embodiments, an amplification oligomer is provided. The amplification oligomer generally includes a target hybridization region, for example, configured to specifically hybridize with HBV nucleic acid. Although oligomers of different lengths and base compositions can be used to amplify HBV nucleic acid, in some embodiments, the oligomers in the present disclosure have a target hybridization region of 10 to 60 bases, 14 to 50 bases, or 15 to 40 bases in length. In some embodiments, an initial amplification oligomer with a relatively long target hybridization region (such as about 30-50 nucleotides, for example 35-45) is used, and an amplification oligomer with a shorter target hybridization region (e.g., about 14-35 nucleotides, such as about 15-30nt) is used in a later stage.

[0121] In certain embodiments, the amplification oligomer as described herein is a promoter primer, which also includes a promoter sequence located 5' of the target hybridization sequence and not complementary to the HBV target nucleic acid. For example, in some embodiments of the oligomer combination for amplifying the HBV target region as described herein, the amplification oligomer as described above is a promoter primer, which also includes a promoter sequence 5' of the target hybridization sequence. Alternatively, the amplification oligomer can be a promoter provider comprising a promoter sequence. In a specific embodiment, the promoter sequence is a T7 RNA polymerase promoter sequence, such as a T7 promoter sequence having a sequence shown in SEQ ID NO: 8. In a specific variant, the promoter primer comprises a non-HBV sequence, including a T7 promoter shown in one of SEQ ID NO: 9, 10, 11, 12 or 13. In some embodiments, at least one, for example, two, three or four promoter primers are provided, which include a target hybridization sequence containing a (+)-chain HBV sequence.

[0122] In some embodiments, the amplification oligomer is not a promoter primer or does not include a promoter sequence. For example, in a PCR-based method, the primer is usually not a promoter primer, and in a TMA-based method, at least one primer that is not a promoter primer is usually used (while also using at least one promoter primer). In some embodiments, at least one, for example, two, three or four amplification oligomers that are not promoter primers are provided, which include a target hybridization sequence containing a (-)-chain HBV sequence.

[0123] In some embodiments, a first amplification oligomer is provided which is a reverse amplification oligomer, ie, it is configured to specifically hybridize to a (+) strand HBV nucleic acid; in other words, its target hybridization sequence corresponds to an "antisense" sequence of HBV.

[0124] In some embodiments, the target sequence of the first amplification oligomer comprises position 449 of the HBV genomic nucleic acid (such as SEQ ID NO: 1), for example, positions 448-450, 447-451, 446-452, 445-453, 444-454, 443-455, 442-456, 441-457, 440-458 or 439-459. In some embodiments, the first amplification oligomer comprises a sequence with at most 1 or 2 mismatches relative to SEQ ID NO: 2. In some embodiments, the first amplification oligomer comprises a sequence with at most 1 or 2 mismatches relative to SEQ ID NO: 3. Various embodiments of the first amplification oligomer are disclosed in the above Summary of the Invention, including with respect to its sequence, any of which can be combined within the feasible range of the features discussed above in this section.

[0125] In some embodiments in which a first amplification oligomer is provided, an additional reverse amplification oligomer different from the first amplification oligomer is also provided, which is also configured to specifically hybridize with a (+) strand HBV nucleic acid. This additional amplification oligomer can anneal to a common position with the first amplification oligomer, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 common positions with the first amplification oligomer. In some embodiments, the target sequence of the additional amplification oligomer comprises 449 positions of an HBV genomic nucleic acid (such as SEQ ID NO: 1), for example, 448-450, 447-451, 446-452, 445-453, 444-454, 443-455, 442-456, 441-457, 440-458 or 439-459. In some embodiments, the additional amplification oligomer comprises a sequence having at most 1 or 2 mismatches relative to SEQ ID NO: 2. In some embodiments, the additional amplification oligomer comprises a sequence having at most 1 or 2 mismatches relative to SEQ ID NO: 3. As described in the Examples, the use of such additional amplification oligomers can improve the relative accuracy of HBV nucleic acid quantification despite sequence variation between genotypes. In some embodiments, the first amplification oligomer comprises at least 10 consecutive nucleotides of SEQ ID NO: 2, and the additional amplification oligomer comprises at least 10 consecutive nucleotides of SEQ ID NO: 3. Various embodiments of additional amplification oligomers are disclosed in the above Summary of the Invention, including with respect to their sequences, any of which can be combined within the feasible range of the features discussed above in this section.

[0126] In some embodiments, a second amplification oligomer is provided which is a forward amplification oligomer, ie, it is configured to specifically hybridize to a (-) strand HBV nucleic acid; in other words, its target hybridization sequence corresponds to an "antisense" sequence of HBV.

[0127] In some embodiments, the target sequence of the second amplification oligomer comprises position 386 or 387 of the HBV genomic nucleic acid (such as SEQ ID NO: 1), for example, position 386-387, 385-388, 384-389, 383-390, 382-391, 381-392, 380-393, 379-394, 378-395, 377-396 or 376-397. In some embodiments, the second amplification oligomer comprises a sequence with at most 1 or 2 mismatches relative to SEQ ID NO: 18. In some embodiments, the second amplification oligomer comprises a sequence of SEQ ID NO: 19, 20, 21, 22, or a sequence with at most 1 or 2 mismatches relative thereto. In some embodiments, the second amplification oligomer comprises a target hybridization sequence comprising positions N to 397 of SEQ ID NO: 1, wherein N is 376, 377, 378, 379, 380, 381, 382, ​​383, or 384, or a sequence with at most 1 or 2 mismatches relative thereto. In some embodiments, the second amplification oligomer comprises a target hybridization sequence comprising positions N to 402 of SEQ ID NO: 1, wherein N is 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, or 389, or a sequence with at most 1 or 2 mismatches relative thereto. Various embodiments of the second amplification oligomer are disclosed in the above Summary of the Invention, including with respect to its sequence, any of which can be combined within the feasible range of the features discussed above in this section.

[0128] In some embodiments in which a second amplification oligomer is provided, a further forward amplification oligomer different from the second amplification oligomer is also provided, which is further configured to specifically hybridize with a (-) strand HBV nucleic acid. This further amplification oligomer can anneal to a common position with the second amplification oligomer, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 common positions with the second amplification oligomer. In some embodiments, the target sequence of the additional amplification oligomer comprises position 389 of the HBV genomic nucleic acid (such as SEQ ID NO: 1), for example, positions 388-390, 387-391, 386-392, 385-393, 384-394, 383-395, 382-396, 381-397, 380-398, 379-399, 378-400, 377-401, or 376-402. In some embodiments, the additional amplification oligomer comprises a sequence with at most 1 or 2 mismatches relative to SEQ ID NO: 19. In some embodiments, the additional amplification oligomer comprises a sequence with at most 1 or 2 mismatches relative to SEQ ID NO: 18, 20, 21, or 22. Despite sequence variation between genotypes, the use of such additional amplification oligomers can improve the relative accuracy of HBV nucleic acid quantification. In some embodiments, the second amplification oligomer comprises at least 10 consecutive nucleotides of SEQ ID NO: 18, and the additional amplification oligomer comprises at least 10 consecutive nucleotides of SEQ ID NO: 19. The target hybridization region of the additional amplification oligomer can be longer than the target hybridization region of the second amplification oligomer, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides longer, such as about 4-6 nucleotides, for example 5 nucleotides. Various embodiments of such additional amplification oligomers are disclosed in the above Summary of the Invention, including with respect to their sequences, any of which can be combined within the feasible range of the features discussed above in this section.

[0129] The first, second and optional additional amplification oligomers discussed above can be used in an amplification reaction to produce a first amplicon, which, for example, contains positions such as 425, for example 420-430, 416-434, 406-444, 396-454, 386-464 or 376-474 of an HBV nucleic acid (such as SEQ ID NO: 1).

[0130] In some embodiments, a third amplification oligomer is provided which is a reverse amplification oligomer, ie, it is configured to specifically hybridize to the (+) strand HBV nucleic acid; in other words, its target hybridization sequence corresponds to the "antisense" sequence of HBV.

[0131] In some embodiments, the target sequence of the third amplification oligomer comprises 698 or 699 of the HBV genomic nucleic acid (such as SEQ ID NO: 1), for example, 698-699, 697-700, 696-701, 695-702, 694-703, 693-704, 692-705, 691-706, 690-707 or 689-708. In some embodiments, the third amplification oligomer comprises a sequence with at most 1 or 2 mismatches relative to SEQ ID NO: 41. Various embodiments of the third amplification oligomer are disclosed in the above Summary of the Invention, including with respect to its sequence, any of which can be combined within the feasible range of the features discussed above in this section.

[0132] In some embodiments, a fourth amplification oligomer is provided which is a forward amplification oligomer, ie, it is configured to specifically hybridize to a (-) strand HBV nucleic acid; in other words, its target hybridization sequence corresponds to an "antisense" sequence of HBV.

[0133] In some embodiments, the target sequence of the fourth amplification oligomer comprises position 656 of the HBV genomic nucleic acid (such as SEQ ID NO: 1), for example, positions 655-657, 654-658, 653-659, 652-660, 651-661, 650-662, 649-663, 648-664, 647-665, or 646-666. In some embodiments, the fourth amplification oligomer comprises a sequence having at most 1 or 2 mismatches relative to SEQ ID NO: 40. In some embodiments, the fourth amplification oligomer comprises a target hybridization sequence comprising positions N to 666 of the HBV nucleic acid sequence (such as SEQ ID NO: 1), wherein N is 646, 647, 648, 649, 650, 651, 652, or 653, or a sequence having at most 1 or 2 mismatches relative thereto. Various embodiments of the fourth amplification oligomer are disclosed in the above Summary of the Invention, including with respect to its sequence, any of which may be combined within the feasible scope of the features discussed above in this section.

[0134] The third and fourth amplification oligomers discussed above can be used in an amplification reaction to produce a second amplicon, which, for example, comprises positions such as 677 or 678, for example 673-682, 668-687, 658-697, 648-707 or 646-708 of an HBV nucleic acid sequence (such as SEQ ID NO: 1).

[0135] In some embodiments, a fifth amplification oligomer is provided which is a forward amplification oligomer, ie, it is configured to specifically hybridize to a (-) strand HBV nucleic acid; in other words, its target hybridization sequence corresponds to an "antisense" sequence of HBV.

[0136] In some embodiments, the target sequence of the fifth amplification oligomer comprises position 264 or 265 of the HBV genomic nucleic acid (such as SEQ ID NO: 1), for example, positions 263-266, 258-271, 255-274, or 250-279. In some embodiments, the fifth amplification oligomer comprises a sequence having at most 1 or 2 mismatches relative to SEQ ID NO: 69. In some embodiments, the fifth amplification oligomer comprises a target hybridization sequence comprising positions N to 279 of the HBV nucleic acid sequence (such as SEQ ID NO: 1), wherein N is 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, or 266, or a sequence having at most 1 or 2 mismatches relative thereto. Various embodiments of the fifth amplification oligomer are disclosed in the above Summary of the Invention, including with respect to its sequence, any of which may be combined within the feasible scope of the features discussed above in this section.

[0137] In some embodiments, a sixth amplification oligomer is provided which is a forward amplification oligomer, ie, it is configured to specifically hybridize to a (-) strand HBV nucleic acid; in other words, its target hybridization sequence corresponds to the "sense" sequence of HBV.

[0138] In some embodiments, the target sequence of the sixth amplification oligomer comprises 461 or 462 of the HBV genomic nucleic acid (such as SEQ ID NO: 1), for example, 461-462, 457-466, 455-468 or 452-471. In some embodiments, the sixth amplification oligomer comprises a sequence with at most 1 or 2 mismatches relative to SEQ ID NO: 73. Various embodiments of the sixth amplification oligomer are disclosed in the above Summary of the Invention, including with respect to its sequence, any of which can be combined within the feasible range of the features discussed above in this section.

[0139] The fifth and sixth amplification oligomers discussed above can be used as displacers, for example, they can promote amplicon production by other oligomers by extension from their 3' ends and accompanying strand displacement, making single-stranded templates available for binding by other oligomers. The fifth and sixth amplification oligomers can reduce or avoid the need for a denaturation step and can enhance sensitivity.

[0140] It should be noted that, for example, the presence of the sixth amplification oligomer does not necessarily mean that all of the first, second, third, fourth and fifth amplification oligomers are present. For example, exponential amplification can be implemented in the presence of only the first and second amplification oligomers, and any one or both of the fifth and sixth amplification oligomers can be used as a displacing agent. In addition, linear amplification can be implemented in the presence of, for example, a promoter primer, without any oligomers of opposite orientation. With necessary changes, this description is applicable to other situations in which ordinal numbers are used, for example, the presence of the second capture oligomer does not necessarily mean that the first capture oligomer is present. In some embodiments, the second, fourth and fifth amplification oligomers are promoter primers, so that they can have any of the features of the promoter primers discussed above.

[0141] In some embodiments, at least one initial amplification oligomer is provided, which is different from other amplification oligomers, to the extent that they exist or are used, such as the second, fourth and any other amplification oligomers. In some embodiments, an initial amplification oligomer is provided, which anneals to one or more common positions with the second amplification oligomer, and has a target hybridization region longer than at least one or two other amplification oligomers (such as the second amplification oligomer and an optional other forward amplification oligomer). In some embodiments, an initial amplification oligomer is provided, which anneals to one or more common positions with the fourth amplification oligomer, and has a target hybridization region longer than the fourth amplification oligomer. As described in the embodiments, it is found that the subsequent amplification and quantification of certain HBV genotypes can be improved using the initial amplification oligomer comprising a long target hybridization region, thereby improving overall detection and quantitative performance. An initial amplification oligomer annealed to one or more common positions with the second amplification oligomer and an initial amplification oligomer annealed to one or more common positions with the fourth amplification oligomer can be provided together. In some embodiments, the initial amplification oligomer is in a composition with one or more target capture oligomers, such as those described below or in the Summary of the Invention above.

[0142] In some embodiments, the target sequence of the initial amplification oligomer that anneals to one or more common positions with the second amplification oligomer comprises position 390 or 391 of the HBV genomic nucleic acid (such as SEQ ID NO: 1), for example, positions 390-391, 389-392, 388-393, 387-394, 386-395, 385-396, 384-397, 383-398, 382-399, 381-400, 380-401, 379-402, 378-403, 377-404, or 376-405. In some embodiments, the initial amplification oligomer comprises a sequence with at most 1 or 2 mismatches relative to SEQ ID NO: 20. In some embodiments, the initial amplification oligomer comprises a sequence of SEQ ID NO: 16 or 17, or a sequence with at most 1 or 2 mismatches relative thereto. In some embodiments, the initial amplification oligomer comprises a target hybridization sequence comprising position N-405 of an HBV genomic sequence (such as SEQ ID NO: 1), wherein N is 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391 or 392, or a sequence with at most 1 or 2 mismatches relative thereto. Various embodiments of the initial amplification oligomer are disclosed in the above Summary of the Invention, including with respect to its sequence, any of which can be combined within the feasible range of the features discussed above in this section. The initial amplification oligomer may also have the features described above in the Summary of the Invention with respect to the second amplification oligomer. For example, the initial amplification oligomer can comprise at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 consecutive nucleotides of SEQ ID NO: 20, or the sequence of SEQ ID NO: 20. In some embodiments, the initial amplification oligomer is a promoter-primer.

[0143] In some embodiments, the target sequence of the initial amplification oligomer that anneals to one or more common positions with the fourth amplification oligomer comprises position 659 of the HBV genomic nucleic acid (such as SEQ ID NO: 1), for example, positions 658-660, 657-661, 656-662, 655-663, 654-664, 653-665, 652-666, 651-667, 650-668, 649-670, 648-671 or 646-672. In some embodiments, the initial amplification oligomer comprises a sequence with up to 1 or 2 mismatches relative to SEQ ID NO: 35, optionally wherein the inosine at the 6th nucleotide from the 3' end of SEQ ID NO: 35 is not matched. In some embodiments, the initial amplification oligomer comprises a sequence of SEQ ID NO: 33 or 31, or a sequence with at most 1 or 2 mismatches thereto, optionally wherein the inosine at the 6th nucleotide from the 3' end of SEQ ID NO: 33 or 31 is mismatched. In some embodiments, the initial amplification oligomer comprises a target hybridization sequence comprising positions N-672 of an HBV genomic sequence (such as SEQ ID NO: 1), wherein N is 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658 or 659, or a sequence with at most 1 or 2 mismatches thereto, optionally wherein the nucleotide corresponding to position 667 is an inosine. Various embodiments of the initial amplification oligomer are disclosed in the above Summary of the Invention, including with respect to its sequence, any of which can be combined within the feasible range of the features discussed above in this section. The initial amplification oligomer may also have the features described in the Summary of the Invention above with respect to the fourth amplification oligomer or any amplification oligomer comprising a target hybridization region comprising at least 10 consecutive nucleotides of SEQ ID NO: 35. For example, the initial amplification oligomer may comprise at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34 consecutive nucleotides of SEQ ID NO: 35 (optionally including inosine at position 30 of SEQ ID NO: 35), or the sequence of SEQ ID NO: 35. In some embodiments, the initial amplification oligomer is a promoter-primer.

[0144] In some embodiments, at least one probe oligomer is provided.Some embodiments of the detection probe hybridized with the sequence of complementary amplification can be DNA or RNA oligomer, or oligomer containing DNA and RNA nucleotide combination, or oligomer synthesized with modified backbone, for example, oligomer including ribonucleotides substituted with one or more 2'-methoxy groups. The probe for detecting the amplified HBV sequence can be unlabeled and detect indirectly (for example, by making another binding partner be combined with the part on the probe) or can be marked with various detectable labels. The detection probe oligomer can contain 2'-methoxy backbone at one or more joints in the nucleic acid backbone.

[0145] In some embodiments, according to the detection probe oligomer disclosed herein, also include labeling. Particularly suitable labeling includes compounds that emit detectable light signals, such as fluorophores or luminescent (e.g., chemiluminescent) compounds that can be detected in a homogeneous mixture. There can be more than one labeling and more than one type of labeling on a particular probe, or detection can rely on a mixture of probes, wherein each probe is labeled with a compound that produces a detectable signal (see, e.g., U.S. Patent Nos. 6,180,340 and 6,350,579, each incorporated herein by reference). Labeling can be attached to the probe by various means, including covalent attachment, chelation, and ionic interactions, but in some embodiments, labeling is covalently attached. For example, in some embodiments, the detection probe has a chemiluminescent label attached, such as, for example, an acridinium ester (AE) compound (see, e.g., U.S. Pat. Nos. 5,185,439; 5,639,604; 5,585,481; and 5,656,744; each of which is incorporated herein by reference), which, in a typical variation, is attached to the probe via a non-nucleotide linker (see, e.g., U.S. Pat. Nos. 5,585,481; 5,656,744; and 5,639,604, each of which is incorporated herein by reference).

[0146] Detection probe oligomers according to the present disclosure may also include non-target hybridization sequences. In some applications, it is necessary to exhibit at least a certain degree of self-complementarity in order to facilitate detection of probes in test samples: target duplexes without first removing unhybridized probes prior to detection. Specific embodiments of such detection probes include, for example, probes that form a conformation maintained by intramolecular hybridization, such as conformations commonly referred to as hairpins. Particularly suitable hairpin probes include "molecular torches" (see, for example, U.S. Patent Nos. 6,849,412; 6,835,542; 6,534,274; and 6,361,945, each incorporated herein by reference) and "molecular beacons" (see, for example, Tyagi et al., supra; U.S. Patent No. 5,118,801 and U.S. Patent No. 5,312,728, supra). In yet other embodiments, the detection probe is a linear oligomer that does not substantially form a conformation maintained by intramolecular bonds.

[0147] For example, a structure called a "molecular beacon" comprises a nucleic acid molecule having a target complementary sequence, an affinity pair (or nucleic acid arm) that holds the probe in a closed conformation in the absence of a target nucleic acid sequence, and a label pair that interacts when the probe is in a closed conformation. The hybridization of the target nucleic acid and the target complementary sequence separates the members of the affinity pair, thereby converting the probe into an open conformation. Due to the reduced interaction of the label pair, the transition to the open conformation can be detected, and the label pair can be, for example, a fluorophore and a quencher (e.g., DABCYL and EDANS). Molecular beacons are fully described in U.S. Patent No. 5,925,517, the disclosure of which is incorporated herein by reference. Molecular beacons that can be used to detect HBV-specific nucleic acid sequences can be generated by attaching to either end of one of the probe (e.g., target hybridization) sequences disclosed herein, a first nucleic acid arm comprising a fluorophore and a second nucleic acid arm comprising a quencher portion. In this configuration, the HBV-specific probe sequence disclosed herein is used as the target complementary "loop" portion of the resulting molecular beacon, and the self-complementary "arm" of the probe represents the "stem" portion of the probe.

[0148] Another example of a self-complementary hybridization assay probe that can be used in conjunction with the present disclosure is a structure commonly referred to as a "molecular torch" (sometimes simply referred to as a torch). These self-reporting probes are designed to include different regions of self-complementarity (creating a "target binding domain" and a "target closing domain") connected by a linker region (e.g., a -(CH2)9- linker) and hybridize to each other under predetermined hybridization assay conditions. When exposed to an appropriate target or denaturing conditions, the two complementary regions of the molecular torch (which can be fully complementary or partially complementary) melt when the predetermined hybridization assay conditions are restored, leaving the target binding domain available for hybridization with the target sequence. The molecular torch is designed so that the target binding domain favors hybridization with the target sequence on the target closing domain. The target binding domain and target closing domain of the molecular torch include interacting labels (e.g., fluorescent / quencher) that are positioned so that a different signal is generated when the molecular torch self-hybridizes, as opposed to when the molecular torch hybridizes with a target nucleic acid, thereby allowing detection of the probe:target duplex in a test sample in the presence of an unhybridized probe having a live label associated therewith. Molecular torches are fully described in US Pat. No. 6,361,945, the disclosure of which is incorporated herein by reference.

[0149] In some embodiments, the molecular torch and the molecular beacon are labeled with a pair of interacting detectable labels. Examples of detectable labels that are members of an interacting label pair include labels that interact with each other through FRET or non-FRET energy transfer mechanisms. Fluorescence resonance energy transfer (FRET) involves the radiationless transfer of energy quanta from an absorption site to its utilization site in a molecule or molecular system, through resonant interactions between chromophores, at distances much greater than the interatomic distance, without the need for conversion to thermal energy, and without the donor and acceptor entering into kinetic collisions. The "donor" is the part that initially absorbs energy, and the "acceptor" is the part to which energy is subsequently transferred. In addition to FRET, there are at least three other "non-FRET" energy transfer processes by which excitation energy can be transferred from the donor to the acceptor molecule.

[0150] When two labels are held in close enough proximity that energy emitted by one label can be received or absorbed by the second label, whether by FRET or non-FRET mechanisms, the two labels are said to be in an "energy transfer relationship" with each other. This is the case, for example, when a molecular beacon is held in a closed state by forming a stem duplex, and fluorescence emission from a fluorophore attached to one arm of the probe is quenched by a quencher moiety on the opposite arm.

[0151] Exemplary labeling moieties of the disclosed molecular torches and molecular beacons include a fluorophore and a second moiety (i.e., a "quencher") having a fluorescence quenching property. In this embodiment, the characteristic signal may be fluorescence of a specific wavelength, but may alternatively be a visible light signal. When fluorescence is involved, in some embodiments, the emission changes due to FRET or radiative energy transfer or a non-FRET mode. When a molecular beacon having a pair of interacting labels in a closed state is stimulated by light of an appropriate frequency, a fluorescent signal is generated at a first level, which may be very low. When the same probe is in an open state and stimulated by light of an appropriate frequency, the fluorophore and quencher moieties are sufficiently separated from each other, substantially eliminating energy transfer between them. Under this condition, the quencher moiety cannot quench fluorescence from the fluorophore moiety. If the fluorophore is stimulated by light energy of an appropriate wavelength, a second level of fluorescent signal higher than the first level will be generated. The difference between the two fluorescence levels is detectable and measurable. Using the fluorophore and quencher moieties in this way, the molecular beacon is "turned on" only in the "open" conformation, and indicates that the probe binds to the target by emitting an easily detectable signal. The conformational state of the probe changes the signal generated by the probe by modulating the interactions between the label moieties.

[0152] Without attempting to distinguish FRET from non-FRET pairs, examples of donor / acceptor label pairs that can be used in conjunction with the present disclosure include fluorescein / tetramethylrhodamine, IAEDANS / fluorescein, EDANS / DABCYL, coumarin / DABCYL, fluorescein / fluorescein, BODIPY FL / BODIPYFL, fluorescein / DABCYL, fluorescein yellow / DABCYL, BODIPY / DABCYL, eosin / DABCYL, erythrosine / DABCYL, tetramethylrhodamine / DABCYL, Texas red / DABCYL, CY5 / BH1, CY5 / BH2, CY3 / BH1, CY3 / BH2, and fluorescein / QSY7 dyes. Those of ordinary skill in the art will appreciate that when donor and acceptor dyes are different, energy transfer can be detected by the appearance of sensitized fluorescence of the acceptor or by quenching the donor fluorescence. When the donor and acceptor species are identical, energy can be detected by the resulting fluorescence depolarization. Non-fluorescent acceptors such as DABCYL and QSY7 dyes advantageously eliminate potential problems with background fluorescence caused by direct (i.e., non-sensitized) acceptor excitation. Exemplary fluorophore moieties that can be used as one member of a donor-acceptor pair include fluorescein, ROX, and CY dyes (such as CY5). Exemplary quencher moieties that can be used as the other member of a donor-acceptor pair include DABCYL and BLACK HOLE QUENCHER moieties, which are available from Biosearch Technologies, Inc., (Novato, Calif.).

[0153] Oligomers that are not intended to be extended by nucleic acid polymerase, such as probe oligomers and capture oligomers, may include a blocker group that replaces the 3'OH to prevent the enzyme-mediated extension of the oligomer in the amplification reaction. For example, in some embodiments, the amplification oligomer and / or the detection probe of the blocking present during the amplification do not have a functional 3'OH, but include one or more blocking groups located at or near the 3' end. In some embodiments, the blocking group near the 3' end is within 5 residues at the 3' end and is large enough to limit the combination of the polymerase and the oligomer, and other embodiments contain a blocking group covalently attached to the 3' end. Many different chemical groups can be used to block the 3' end, such as alkyl, non-nucleotide linker, alkane-diol dideoxynucleotide residues and cordycepin.

[0154] Although the oligonucleotide probes of different lengths and base compositions can be used for detecting HBV nucleic acid, some embodiments of probes in the present disclosure are 10 to 60 bases in length, or 14 to 50 bases in length, or 15 to 30 bases in length. A first probe oligomer can be provided, which is configured to specifically hybridize with the first amplicon discussed above. Alternatively or additionally, a second probe oligomer can be provided, which is configured to specifically hybridize with the first amplicon discussed above.

[0155] In some embodiments, the target sequence of the first probe oligomer comprises position 424 of a HBV genomic nucleic acid (such as SEQ ID NO: 1), for example, positions 423-425, 421-427, 419-429, 417-431, 415-433, 413-435, 411-437, 410-438, 409-439, or 408-440. In some embodiments, the probe oligomer comprises a sequence having at most 1 or 2 mismatches relative to SEQ ID NO: 82. In some embodiments, the probe oligomer comprises a sequence of SEQ ID NO: 29, or a sequence having at most 1 or 2 mismatches relative thereto. Various embodiments of the probe oligomer are disclosed in the above Summary of the Invention, including with respect to its sequence, any of which can be combined within the feasible range of the features discussed above in this section.

[0156] In some embodiments, the target sequence of the second probe oligomer comprises position 675 or 676 of the HBV genomic nucleic acid (such as SEQ ID NO: 1), for example, positions 675-676, 674-677, 673-678, 672-679, 671-680, 670-681, 669-682 or 668-683. In some embodiments, the probe oligomer comprises a sequence having at most 1 or 2 mismatches relative to SEQ ID NO: 83. In some embodiments, the probe oligomer comprises a sequence of SEQ ID NO: 84, 85, 40, or a sequence having at most 1 or 2 mismatches relative thereto. Various embodiments of the probe oligomer are disclosed in the above Summary of the Invention, including with respect to its sequence, any of which can be combined within the feasible range of the features discussed above in this section.

[0157] In some embodiments, at least one capture oligomer is provided, for example, two, three or four capture oligomers. It should be understood that when there are two or more capture oligomers, their target hybridization sequences are different from each other. The one or more capture oligomers contain a target hybridization sequence configured to specifically hybridize with HBV nucleic acid, for example, a length of 10 to 60 bases, or a length of 14 to 50 bases, or a length of 15 to 30 bases. For example, in a specific embodiment of the capture probe, one, two, three or four capture probes have a target hybridization sequence selected from SEQ ID NO: 49, 53, 57, 61 and 96-104. The target hybridization sequence is covalently linked to a sequence or portion bound to a fixed probe, for example an oligomer attached to a solid substrate such as a bead.

[0158] In more specific embodiments, the capture oligomer includes a tail (e.g., a 3' tail) that is not complementary to the HBV target sequence, but specifically hybridizes to the sequence of an immobilized binding partner (e.g., an immobilized probe), thereby serving as a portion that allows the target nucleic acid to be separated from other sample components, such as previously described in, for example, U.S. Patent No. 6,110,678, incorporated herein by reference. Any sequence can be used for the tail region, which is generally about 5 to 50 nt long, and certain embodiments include a substantially homopolymeric tail ("poly-N sequence") of at least about 10 nt, for example, about 10 to 40 nt. (For example, A 10 To A 40 ), such as about 14 to 33 nt (e.g., A 14 To A 30 or T3A 14 To T3A 30), which binds to a complementary immobilized sequence (e.g., poly-T) attached to a solid support (e.g., a matrix or particle). For example, in a specific embodiment comprising a capture probe with a 3' tail, one, two, three or four capture probes have a sequence selected from SEQ ID NOs: 45, 46, 47 and 48.

[0159] In some embodiments, a first capture oligomer is provided. In some embodiments, the target sequence of the first capture oligomer comprises position 719 of a HBV genomic nucleic acid (such as SEQ ID NO: 1), for example, positions 718-720, 716-722, 714-724, 712-726, 710-728, 709-729, 708-730, or 707-731. In some embodiments, the first capture oligomer comprises a sequence having at most 1 or 2 mismatches relative to SEQ ID NO: 49. In some embodiments, the first capture oligomer comprises a sequence having at most 1 or 2 mismatches relative to SEQ ID NO: 99. In some embodiments, the first capture oligomer comprises a sequence having at most 1 or 2 mismatches relative to SEQ ID NO: 45. Various embodiments of the first capture oligomer are disclosed in the Summary of the Invention above, including with respect to its sequence, any of which can be combined within the feasible range of the features discussed above in this section.

[0160] In some embodiments, a second capture oligomer is provided. In some embodiments, the target sequence of the second capture oligomer comprises position 745 of a HBV genomic nucleic acid (such as SEQ ID NO: 1), for example, positions 744-746, 742-748, 740-750, 738-752, 736-754, 735-755, 734-756, or 733-757. In some embodiments, the second capture oligomer comprises a sequence having at most 1 or 2 mismatches relative to SEQ ID NO: 53. In some embodiments, the second capture oligomer comprises a sequence having at most 1 or 2 mismatches relative to SEQ ID NO: 100, 101, or 104. In some embodiments, the second capture oligomer comprises a sequence having at most 1 or 2 mismatches relative to SEQ ID NO: 46. Various embodiments of the second capture oligomer are disclosed in the Summary of the Invention above, including with respect to its sequence, any of which can be combined within the feasible range of the features discussed above in this section.

[0161] In some embodiments, a third capture oligomer is provided. In some embodiments, the target sequence of the third capture oligomer comprises position 1180 or 1181 of a HBV genomic nucleic acid (such as SEQ ID NO: 1), for example, positions 1180-1181, 1178-1183, 1176-1185, 1174-1187, 1172-1189, 1170-1191, 1169-1192, or 1168-1193. In some embodiments, the third capture oligomer comprises a sequence having at most 1 or 2 mismatches relative to SEQ ID NO: 57. In some embodiments, the third capture oligomer comprises a sequence having at most 1 or 2 mismatches relative to SEQ ID NO: 96. In some embodiments, the third capture oligomer comprises a sequence having at most 1 or 2 mismatches relative to SEQ ID NO: 102. In some embodiments, the third capture oligomer comprises a sequence having at most 1 or 2 mismatches relative to SEQ ID NO: 47. Various embodiments of the third capture oligomer are disclosed above in the Summary of the Invention, including with respect to its sequence, any of which may be combined within the feasible range of the features discussed above in this section.

[0162] In some embodiments, a fourth capture oligomer is provided. In some embodiments, the target sequence of the fourth capture oligomer comprises position 1303 of a HBV genomic nucleic acid (such as SEQ ID NO: 1), for example, positions 1302-1304, 1300-1306, 1298-1308, 1296-1310, 1294-1312, 1292-1314, 1291-1315, or 1290-1316. In some embodiments, the fourth capture oligomer comprises a sequence with at most 1 or 2 mismatches relative to SEQ ID NO: 61. In some embodiments, the fourth capture oligomer comprises a sequence with at most 1 or 2 mismatches relative to SEQ ID NO: 97, 98, or 103. In some embodiments, the fourth capture oligomer comprises a sequence with at most 1 or 2 mismatches relative to SEQ ID NO: 48. Various embodiments of the fourth capture oligomer are disclosed in the Summary above, including with respect to its sequence, any of which may be combined within the feasible range of the features discussed above in this section.

[0163] An internal control oligomer can be provided, for example, to confirm that a negative result is valid by establishing conditions suitable for amplification. An exemplary control target capture oligomer is SEQ ID NO: 80. Exemplary control amplification oligomers are SEQ ID NOs: 77 and 78. An exemplary control probe oligomer is SEQ ID NO: 79. A control template that can be amplified by the control amplification oligomer can also be provided. The control template can be prepared according to known protocols. See, for example, U.S. Patent No. 7,785,844, which is incorporated herein by reference and describes an internal control consisting of an in vitro synthesized transcript containing a portion of an HIV-1 sequence and a unique sequence targeted by an internal control probe.

[0164] In certain aspects of the present disclosure, a combination of at least two oligomers is provided for determining the presence or absence of HBV in a sample or for quantifying HBV. In some embodiments, the oligomer combination includes at least two amplification oligomers suitable for amplifying a target region of an HBV target nucleic acid, for example, a sequence having SEQ ID NO: 1 or 105-125, or any HBV isolate or construct mentioned in the examples, or an accession number is provided herein. In such embodiments, at least one amplification oligomer comprises a target hybridization sequence in the sense direction ("sense THS"), and at least one amplification oligomer comprises a target hybridization sequence in the antisense direction ("antisense THS"), wherein the sense THS and the antisense THS are each configured to specifically hybridize with a target sequence within the HBV sequence. It should be understood that the target hybridization sequence is selected so that the HBV sequence targeted by the antisense THS is located downstream of the HBV sequence targeted by the sense THS (i.e., the position of at least two amplification oligomers is such that they are located on the flank of the target region to be amplified).

[0165] The oligomers can be provided in various combinations (e.g., kits or compositions), for example, comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 first amplification oligomers, additional reverse amplification oligomers, second amplification oligomers, additional forward amplification oligomers, third amplification oligomers, fourth amplification oligomers, fifth amplification oligomers, sixth amplification oligomers, an initial amplification oligomer annealed to one or more common locations with the second amplification oligomer, an initial amplification oligomer annealed to one or more common locations with the fourth amplification oligomer, a first probe oligomer, a second probe oligomer, a first capture oligomer, a second capture oligomer, a third capture oligomer, and a fourth capture oligomer. The first capture oligomer and the second capture oligomer optionally further comprise at least one initial amplification oligomer; the first capture oligomer and the third capture oligomer optionally further comprise at least one initial amplification oligomer; the first capture oligomer and the fourth capture oligomer optionally further comprise at least one initial amplification oligomer; the second capture oligomer and the third capture oligomer optionally further comprise at least one initial amplification oligomer; the second capture oligomer and the fourth capture oligomer optionally further comprise at least one initial amplification oligomer; the third capture oligomer and the fourth capture oligomer optionally further comprise at least one initial amplification oligomer; the first amplification oligomer and the second amplification oligomer amplification oligomer, optionally further comprising a first probe oligomer; a first, second and additional reverse amplification oligomer, optionally further comprising a first probe oligomer; a first, second and additional forward amplification oligomer, optionally further comprising a first probe oligomer; a first, second, additional forward and additional reverse amplification oligomer, optionally further comprising a first probe oligomer; an initial amplification oligomer annealed to one or more common positions with the second amplification oligomer, at least one capture oligomer, a first amplification oligomer and a second amplification oligomer, optionally further comprising a first probe oligomer; an initial amplification oligomer annealed to one or more common positions with the second amplification oligomer, at least two capture oligomers, a first amplification oligomer and a second amplification oligomer, Optionally further comprising a first probe oligomer; a third amplification oligomer and a fourth amplification oligomer, optionally further comprising a second probe oligomer; a third amplification oligomer, a fourth amplification oligomer and an initial amplification oligomer annealed to one or more common positions with the fourth amplification oligomer, optionally further comprising a second probe oligomer; a third amplification oligomer, a fourth amplification oligomer, an initial amplification oligomer annealed to one or more common positions with the fourth amplification oligomer, and at least one capture oligomer, optionally further comprising a second probe oligomer; an initial amplification oligomer annealed to one or more common positions with the second amplification oligomer, at least two capture oligomers, a first amplification oligomer and a second amplification oligomer, optionally further comprising a first probe oligomer;An initial amplification oligomer annealed to one or more common positions with a second amplification oligomer, at least one capture oligomer, a first amplification oligomer, a second amplification oligomer, a third amplification oligomer, and a fourth amplification oligomer, optionally further comprising a third capture oligomer and / or a fourth capture oligomer; or an initial amplification oligomer annealed to one or more common positions with a second amplification oligomer, an initial amplification oligomer annealed to one or more common positions with a fourth amplification oligomer, at least one capture oligomer, a first amplification oligomer, a second amplification oligomer, a third amplification oligomer, and a fourth amplification oligomer, optionally further comprising a first and a second probe oligomer. The combination may also include a control oligomer or a combination thereof, for example, two control amplification oligomers, a control target capture oligomer, and / or a control probe oligomer. In some embodiments, there are both a first and a second amplification oligomer. In some embodiments, there are both an initial and a third amplification oligomer. ;

[0166] In some embodiments, the combination does not contain more than 17, 16, 15, 14, 13, 12, 11, 10 or 9 different oligomers, excluding control oligomers. In such embodiments, the variant is present in trace amounts (e.g., relative to the main species of the oligomer, such as the oligomer with the most similar sequence to the variant, about 15 mol% or less, or about 10 mol% or less), such as, the result of misincorporation, double incorporation, omission or other errors during oligomer synthesis is not considered to be a significant oligomer.

[0167] In some embodiments, a combination of oligomers is provided as described below in any of the Examples or in a single reaction described in the Examples.

[0168] In some embodiments, for example, in a kit or composition, the combination of oligonucleotides is configured to specifically hybridize with nucleic acids of at least three, four, five, six, seven, eight or nine HBV genotypes (e.g., A, B, C, C2, D, E, F, G or H), optionally with minimal cross-reactivity with other non-HBV nucleic acids suspected to be present in the sample (e.g., other blood-borne pathogens, such as one or more or all of the microorganisms and / or blood-borne viruses listed in Table 6). In some embodiments, the combination of oligomers can be used to quantify such strains within 1 log of HBV A. In some embodiments, the combination of oligomers can be used to quantify such strains within 0.5 log of HBV A. In some aspects, the compositions of the present disclosure are configured to specifically hybridize with HBV nucleic acids, with minimal cross-reactivity with one or more or all of hepatitis A, rubella, hepatitis C, herpes simplex 1, herpes simplex 2, HIV2, parvovirus, dengue, CMV, HTLV, Epstein-Barr and West Nile virus. In some embodiments, the compositions of the present disclosure are configured to specifically hybridize to HBV nucleic acid with minimal cross-reactivity to one or more or all of Candida albicans, Propionibacterium acnes, Staphylococcus aureus, Staphylococcus epidermidis, or Neisseria gonorrhoeae. In one aspect, the compositions of the present disclosure are part of a multiplex system that also includes components and methods for detecting one or more of these organisms.

[0169] The present disclosure also provides a reaction mixture for determining the presence or absence of HBV target nucleic acid in a sample or quantifying its amount. The reaction mixture according to the present disclosure includes at least one or more of the following: an oligomer combination as described herein, for amplifying HBV target nucleic acid; a capture probe oligomer as described herein, for purifying HBV target nucleic acid; and a detection probe oligomer as described herein, for determining the presence or absence of HBV amplification products. In some embodiments, any of the above oligomer combinations are present in the reaction mixture. The reaction mixture may also include many optional components, such as capture probe nucleic acid arrays. For amplification reaction mixtures, the reaction mixture generally includes other reagents suitable for implementing in vitro amplification, such as buffers, saline solutions, suitable triphosphate nucleotides (e.g., dATP, dCTP, dGTP, dTTP, ATP, CTP, GTP and UTP) and / or enzymes (e.g., reverse transcriptase and / or RNA polymerase), and generally includes a test sample component, in which HBV target nucleic acid may be present or absent. In addition, for a reaction mixture comprising detection probes and amplification oligomer combinations, the detection probe oligomers and amplification oligomers used in the reaction mixture are selected to be connected by a common target region (i.e., the reaction mixture will include probes that bind to sequences that can be amplified by the amplification oligomer combination of the reaction mixture).

[0170] The present disclosure also provides a kit for practicing the methods described herein. The kit according to the present disclosure comprises at least one or more of the following: an amplification oligomer combination as described herein for amplifying HBV target nucleic acid; at least one capture probe oligomer as described herein for purifying HBV target nucleic acid; and at least one detection probe oligomer as described herein for determining the presence or absence of HBV amplification products. In some embodiments, any of the above oligomer combinations are present in the kit. The kit may also include a number of optional components, such as capture probe nucleic acid arrays. Other reagents that may be present in the kit include reagents suitable for implementing in vitro amplification, such as buffers, saline solutions, suitable triphosphate nucleotides (e.g., dATP, dCTP, dGTP, dTTP, ATP, CTP, GTP, and UTP), and / or enzymes (e.g., reverse transcriptase and / or RNA polymerase). The oligomers as described herein can be packaged in a variety of different embodiments, and those skilled in the art will appreciate that the present disclosure includes many different kit configurations. For example, the kit may include an amplification oligomer for only one target region of the HBV genome, or it may include an amplification oligomer for multiple HBV target regions, such as for generating the first and second amplicons as described above. In addition, for kits that include detection probes and amplification oligomer combinations, the selection of amplification oligomers and detection probe oligomers of the kit are connected by a common target region (i.e., the kit will include a probe that binds to a sequence that can be amplified by the amplification oligomer combination of the kit). In certain embodiments, the kit also includes a set of instructions for practicing the methods according to the present disclosure, wherein the instructions can be associated with the package insert and / or the packaging of the kit or its components.

[0171] C. Methods and Uses

[0172] Any method disclosed herein should also be understood to involve the disclosure of the corresponding use of the materials involved in the method for the purpose of the method. Any oligomer comprising an HBV sequence and any combination (e.g., kits and compositions) comprising such an oligomer should be understood to also be disclosed for detecting or quantifying HBV, and for preparing a composition for detecting or quantifying HBV. .

[0173] Broadly speaking, the method may include one or more of the following components: target capture, in which HBV nucleic acid is annealed to a capture oligomer and optionally to an initial amplification oligomer; separation, such as washing, to remove substances unrelated to the capture oligomer; linear amplification; exponential amplification; and amplicon detection, such as amplicon quantification, which can be implemented in real time with exponential amplification. Certain embodiments relate to each of the foregoing steps. Certain embodiments relate to exponential amplification without linear amplification. Certain embodiments relate to washing, separation, and linear amplification. Certain embodiments relate to exponential amplification and amplicon detection. Certain embodiments relate to any two components listed above. Certain embodiments relate to any two components listed adjacent to each other above, such as washing and linear amplification, or linear amplification and exponential amplification.

[0174] In some embodiments, amplification includes contacting the sample with at least two oligomers for amplifying a target region of HBV nucleic acid corresponding to a HBV target nucleic acid, wherein the oligomers include at least two amplification oligomers as described above (e.g., one or more oriented in the sense direction and one or more oriented in the antisense direction for exponential amplification); (2) performing an in vitro nucleic acid amplification reaction, wherein any HBV target nucleic acid present in the sample is used as a template for producing an amplification product; and (3) detecting the presence or absence of the amplification product, thereby determining the presence or absence of HBV in the sample, or quantifying the amount of HBV nucleic acid in the sample.

[0175] In some embodiments, amplification includes contacting the sample with at least four oligomers for amplifying a HBV nucleic acid target region corresponding to an HBV target nucleic acid, wherein the oligomers include at least two amplification oligomers for producing a first amplicon as described above (e.g., one or more oriented in a sense direction and one or more oriented in an antisense direction for exponential amplification) and at least two amplification oligomers for producing a second amplicon as described above (e.g., one or more oriented in a sense direction and one or more oriented in an antisense direction for exponential amplification); (2) performing an in vitro nucleic acid amplification reaction, wherein any HBV target nucleic acid present in the sample is used as a template for producing an amplification product; and (3) detecting the presence or absence of the first or second amplicon, thereby determining the presence or absence of HBV in the sample, or quantifying the amount of HBV nucleic acid in the sample.

[0176] Detection methods according to the present disclosure may also include the step of obtaining a sample to be subjected to subsequent steps of the method. In certain embodiments, "obtaining" a sample to be used includes, for example, receiving a sample at a testing facility or other location where one or more steps of the method are performed, and / or recovering a sample from a location within a facility where one or more steps of the method are performed (e.g., from storage or other storage).

[0177] In certain embodiments, the method further comprises, for example, prior to amplification, such as prior to the capture step, purifying the HBV target nucleic acid from other components of the sample. This purification may include separating and / or concentrating the organism contained in the sample from other sample components, or removing or degrading non-nucleic acid sample components (e.g., proteins, carbohydrates, salts, lipids, etc.). In some embodiments, the DNA in the sample is degraded, for example, with a DNA enzyme, and optionally removing or inactivating the DNA enzyme or removing degraded DNA.

[0178] In a specific embodiment, purifying the target nucleic acid includes capturing the target nucleic acid so as to specifically or non-specifically separate the target nucleic acid from other sample components. Non-specific target capture methods may involve selective precipitation of nucleic acids from a substantially aqueous mixture, adhering the nucleic acids to a support, washing the support to remove other sample components, or other means of physically separating nucleic acids from a mixture containing HBV nucleic acid and other sample components.

[0179] Target capture typically occurs in a solution phase mixture containing one or more capture probe oligomers that specifically hybridize to the HBV target sequence under hybridization conditions, typically at a temperature above the T of the tail sequence:immobilized probe sequence duplex. m For embodiments comprising a capture probe tail, the HBV-target:capture-probe complex is captured by adjusting the hybridization conditions so that the capture probe tail hybridizes with the immobilized probe. Certain embodiments use a particulate solid support, such as paramagnetic beads.

[0180] Separation can be performed after capture, wherein the complex on the solid support is separated from other sample components. Separation can be completed by any appropriate technique, for example, washing the support associated with the HBV-target sequence once or multiple times (for example, 2 or 3 times) to remove other sample components and / or unbound oligomers. In an embodiment using a granular solid support (such as paramagnetic beads), the particles associated with the HBV-target can be suspended in a washing solution and recovered from the washing solution. In some embodiments, by using magnetic attraction. In order to limit the number of processing steps, the HBV target nucleic acid can be amplified by simply mixing the HBV target sequence in the complex on the support with an amplification oligomer and performing an amplification step.

[0181] Linear amplification can be implemented, for example, by contacting the target nucleic acid sequence with a first-stage amplification reaction mixture, which supports the linear amplification of the target nucleic acid sequence and lacks at least one component required for its exponential amplification. In some embodiments, the first-stage amplification reaction mixture includes an amplification enzyme selected from a reverse transcriptase, a polymerase, and a combination thereof. The polymerase is typically selected from RNA-dependent DNA polymerase, DNA-dependent DNA polymerase, DNA-dependent RNA polymerase, and a combination thereof. In some embodiments, the first-stage amplification reaction mixture also includes a ribonuclease (RNase), such as ribonuclease H or a reverse transcriptase with ribonuclease H activity. In some embodiments, the first-stage amplification mixture includes a reverse transcriptase with ribonuclease H activity and an RNA polymerase.

[0182] In some embodiments, the first stage amplification mixture may also include an amplification oligonucleotide. The amplification oligonucleotide may include a 5' promoter sequence for an RNA polymerase, such as T7 RNA polymerase, and / or a blocked 3' end that prevents its enzymatic extension. In addition, the first stage amplification mixture may sometimes include a blocker oligonucleotide to prevent the enzymatic extension of the target nucleic acid sequence from exceeding the desired end point.

[0183] As described above, the key feature of the first stage amplification reaction is that it cannot support an exponential amplification reaction because one or more components required for exponential amplification are lacking, and / or reagents that inhibit exponential amplification are present, and / or the temperature of the reaction mixture is not conducive to exponential amplification, etc. Without limitation, the lack of components and / or inhibitors and / or reaction conditions required for exponential amplification can be selected from the following group: an amplification oligonucleotide (e.g., an amplification oligonucleotide comprising a 5' promoter sequence of an RNA polymerase, a non-promoter amplification oligonucleotide, or a combination thereof), an enzyme (e.g., a polymerase, such as an RNA polymerase), a nuclease (e.g., an exonuclease, an endonuclease, a lyase, a ribonuclease, a phosphorylase, a glycosylase, etc.), an enzyme cofactor, a chelating agent (e.g., EDTA or EGTA), ribonucleotide triphosphates (rNTPs), deoxyribonucleotide triphosphates (dNTPs), Mg, 2+ , salts, buffers, enzyme inhibitors, blocking oligonucleotides, pH, temperature, salt concentration and combinations thereof. In some cases, the lack of a component may be indirectly involved, such as an agent that reverses the effect of an inhibitor of exponential amplification present in the first phase reaction.

[0184] Exponential amplification of HBV target sequences utilizes an in vitro amplification reaction that uses at least two amplification oligomers that flank the target region to be amplified.

[0185] In some embodiments, at least a first and a second amplification oligomer as described above are provided. In a specific embodiment, the target region to be amplified corresponds substantially to the region of SEQ ID NO: 1, including nucleotides 417 or 418, e.g., about 415-420, 410-425, 405-430, 400-435, 395-440, 390-445, 385-450, 380-455 or 376-459 (including the oligomer sequence incorporated into the amplification product).

[0186] In some embodiments, at least a third and a fourth amplification oligomer as described above are provided. In a specific embodiment, the target region to be amplified corresponds substantially to the region of SEQ ID NO: 1, including nucleotide 677, e.g., about 675-679, 670-684, 665-689, 660-694, 655-699, 650-704 or 646-708 (including the oligomer sequence incorporated into the amplification product).

[0187] The particularly suitable amplification oligomer combination for amplifying these target regions is as described above. In some embodiments, the target region flanked by the first and second amplification oligomers and the third and fourth amplification oligomers is amplified in the same reaction mixture. Suitable amplification methods include, for example, replicase-mediated amplification, polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA) and transcription-mediated or transcription-related amplification (TMA).

[0188] For example, some amplification methods using TMA amplification include the following steps. In short, a target nucleic acid containing a sequence to be amplified is provided as a single-stranded nucleic acid (e.g., ssRNA, such as HBV RNA). It will be appreciated by those skilled in the art that, alternatively, DNA can be used for TMA; conventional melting of double-stranded nucleic acids (e.g., dsDNA) can be used to provide single-stranded target nucleic acids. A promoter primer (e.g., a third amplification oligomer comprising a promoter as described above) specifically binds to the target nucleic acid at its target sequence, and a reverse transcriptase (RT) uses the target chain as a template to extend the 3' end of the promoter primer to produce a cDNA extension product, and if ssRNA is the original template, an RNA: DNA duplex is produced. A ribonuclease digests the RNA chain of the RNA: DNA duplex, and a second primer specifically binds to its target sequence, which is located on the cDNA chain downstream of the promoter primer end. RT synthesizes a new DNA chain by extending the 3' end of another primer using the first cDNA template to produce a dsDNA containing a functional promoter sequence. Then, an RNA polymerase specific for the promoter sequence initiates transcription to produce RNA transcripts, which are about 100 to 1000 amplified copies ("amplicons") of the initial target strand in the reaction. Amplification continues when another primer specifically binds to the target sequence in each amplicon, and RT produces DNA copies from the amplicon RNA template to produce an RNA: DNA duplex. Ribonucleases in the reaction mixture digest the amplicon RNA from the RNA: DNA duplex, and the promoter primer specifically binds to the complementary sequence in the newly synthesized DNA. RT extends the 3' end of the promoter primer to produce dsDNA, which contains a functional promoter bound by RNA polymerase to transcribe additional amplicons complementary to the target strand. The autocatalytic cycle of preparing more copies of the amplicon is repeated during the reaction, resulting in an amplification of the target nucleic acid present in the sample by about 1 billion times. The amplification products can be detected in real time during amplification or at the end of the amplification reaction by using probes that specifically bind to the target sequence contained in the amplification products. Detection of the signal generated by the bound probe indicates the presence of the target nucleic acid in the sample.

[0189] In some embodiments, the method utilizes "reverse" TMA reaction. In these variations, the initial or "forward" amplification oligomer is an oligonucleotide that initiates hybridization with the target nucleic acid near the 3'-end of the target region. Reverse transcriptase (RT) synthesizes the cDNA chain by using the target nucleic acid as the 3'-end of the template extension primer. Another or "reverse" amplification oligomer is a promoter primer or promoter provider, which has a target hybridization sequence that is configured to hybridize with the target sequence included in the synthesized cDNA chain. When the second amplification oligomer is a promoter primer, RT uses the cDNA chain as a template to extend the 3' end of the promoter primer to produce a second cDNA copy of the target sequence chain, thereby producing a dsDNA containing a functional promoter sequence. Then, continue to amplify as described in the previous paragraph, for transcription from the promoter sequence using RNA polymerase. Or, when the second amplification oligomer is a promoter provider, the terminator oligonucleotide that is usually hybridized with the target sequence near the 5'-end of the target region is used to terminate the extension of the initiator oligomer at the 3'-end of the terminator oligonucleotide, thereby providing a definite 3'-end by extending the synthetic initial cDNA chain from the initiator oligomer. Then, the target hybridization sequence of the promoter provider is hybridized with the definite 3'-end of the initial cDNA chain, and the 3'-end of the cDNA chain is extended to add a sequence complementary to the promoter sequence of the promoter provider, resulting in the formation of a double-stranded promoter sequence. Then, using the RNA polymerase that recognizes the double-stranded promoter and begins to transcribe therefrom, the initial cDNA chain is used for template transcription and a plurality of RNA transcripts complementary to the initial cDNA chain, excluding the promoter portion. Then, each of these RNA transcripts can be used as a template for further amplification from the first initiator amplification oligomer.

[0190] Any of a variety of known techniques can be used to implement the detection step, to detect the signal specifically related to the target sequence of amplification, such as by hybridizing the amplified product with the detection probe of the mark and detecting the signal produced by the probe of the mark. The detection step can also provide other information about the sequence of amplification, such as all or part of its nucleic acid base sequence. Detection can be implemented after the amplification reaction is completed, or detection can be implemented simultaneously with the amplification target area, such as, in real time. In one embodiment, the detection step allows homogeneous detection, such as detecting hybridized probes without removing unhybridized probes from the mixture (see, for example, U.S. Patent Nos. 5,639,604 and 5,283,174, each incorporated herein by reference). In some embodiments, nucleic acid is related to the surface causing physical changes, such as detectable electrical changes. The amplified nucleic acid can be detected as follows: by concentrating them in a matrix or on a matrix and detecting nucleic acid or dyes (for example, intercalators, such as ethidium bromide or reticular green) associated with them, or detecting the increase of dyes associated with nucleic acid in a solution phase. Other detection methods can use nucleic acid detection probes that are configured to specifically hybridize with sequences in the amplification product and detect the presence of the probe: product complex, or by using a probe complex that can amplify a detectable signal associated with the amplification product (e.g., U.S. Patent Nos. 5,424,413; 5,451,503; and 5,849,481; each incorporated herein by reference). Directly or indirectly labeled probes that specifically bind to the amplification product provide a detectable signal that indicates the presence of the target nucleic acid in the sample. In particular, the amplification product will contain the target sequence or be complementary to a sequence in the HBV genomic RNA, and the probe will bind directly or indirectly to a sequence contained in the amplification product to indicate the presence of HBV nucleic acid in the test sample.

[0191] In an embodiment where the amplification product is detected near or at the end of the amplification step, a linear detection probe can be used to provide a signal to indicate the hybridization of the probe with the amplification product. An example of such detection uses a luminescent labeled probe that hybridizes with the target nucleic acid. Then, the luminescent label is hydrolyzed from the non-hybridized probe. Using a luminometer, detection is implemented by chemiluminescence (see, for example, International Patent Application Publication No. WO 89 / 002476, incorporated herein by reference). In other embodiments using real-time detection, the detection probe can be a hairpin probe, such as a molecular beacon, a molecular torch or a hybridization switch probe, which is labeled with a reporter portion, and the reporter portion is detected when the probe is combined with the amplification product. Such probes may include target hybridization sequences and non-target hybridization sequences. Various forms of such probes have been described previously (see, e.g., U.S. Patent Nos. 5,118,801; 5,312,728; 5,925,517; 6,150,097; 6,849,412; 6,835,542; 6,534,274; and 6,361,945; and U.S. Patent Application Publication Nos. 20060068417A1 and 20060194240A1; each incorporated herein by reference).

[0192] In some embodiments, a molecular torch (sometimes simply referred to as a torch) is used for detection. In some embodiments, the torch is a probe oligomer as disclosed above.

[0193] Generally, the disclosed methods can include the step of consulting a standard curve relating pre-amplified amounts of the analyte polynucleotide and post-amplified amounts of the analyte amplicons.

[0194] Since the real-time amplification reaction advantageously has a quantitative relationship between the number of analyte polynucleotides input into the reaction and the number of analyte amplicons synthesized as a function of time, the number of analyte polynucleotides present in the test sample can be determined using a standard curve. For example, a plurality of amplification reactions containing a known amount of polynucleotide standards can be run in parallel with an amplification reaction prepared using a test sample containing an unknown amount of analyte polynucleotides. Alternatively, a standard curve can be prepared in advance so that the curve does not have to be prepared each time the analysis procedure is performed. This curve prepared in advance can even be stored electronically in the storage device of the test instrument. Subsequently, a standard curve with a pre-amplification amount of polynucleotide standards on the first axis and some marks of the time required for a certain level of nucleic acid amplification on the second axis are prepared (e.g., emergence time above background signal). Then, the amount of the analyte amplicon measured by the test reaction after amplification is positioned on the amplification rear axis of the standard curve. The corresponding value on the other axis of the curve represents the pre-amplification amount of the analyte polynucleotides present in the test reaction. Therefore, by consulting the standard curve, the number of molecules of the analyte polynucleotides present in the test sample is determined, or more specifically, by comparing the quantitative results obtained by the test sample with the standard curve, which is a program familiar to those of ordinary skill in the art.

[0195] Program described herein can be easily used for the analyte polynucleotide (for example, HBV nucleic acid) present in quantitative test sample.In fact, if a plurality of standard control amplification reactions are started using a known number of analyte polynucleotide standards, and if a test reaction including an unknown number of analyte polynucleotide molecules is carried out, then after the time required for measurement, it becomes possible to realize a certain level of amplification in each reaction to determine the quantity of analyte polynucleotide molecules that must already exist in the test sample.The relationship between the quantity of analyte polynucleotide molecules input to standard amplification reaction and the time required for realizing a certain level of amplification is conveniently established using a chart.Determine the quantity of analyte polynucleotide molecules present in the test sample, it is only a problem of determining the quantity of analyte polynucleotide molecules corresponding to the analyte amplicon signal intensity measured from a standard diagram.This illustrates how analyte polynucleotide standards are used in combination with polynucleotide amplification reactions, with the pre-amplification amount of the analyte polynucleotide contained in the quantitative test sample.

[0196] In some embodiments, quantitative analysis is performed based on one or both of the first and second amplicons, such as those discussed above. In some embodiments, for example, the levels of the first and second amplicons are determined based on signals from the first and second probe oligomers (e.g., labeled probe oligomers), and the first and second probe oligomers are configured to specifically hybridize with the first and second amplicons, respectively. The level can be compared with a predetermined threshold value to determine how to determine the level of HBV nucleic acid in the sample. For example, if one or two levels are approximately equal to or higher than a predetermined threshold value, a higher level can be used to determine the level of HBV nucleic acid in the sample. On the other hand, if one or two levels are lower than a predetermined threshold value, the mean value of the level can be used to determine the level of HBV nucleic acid in the sample. In some embodiments, the mean value is an arithmetic mean (e.g., 20 copies / ml based on 10 and 30 copies / ml of the first and second levels). In some embodiments, the mean value is a geometric mean (e.g., 1.2log copies / ml based on 1.1 and 1.3log copies / ml of the first and second levels).

[0197] Level can be expressed in various ways, such as concentration, absolute number of copies, quality, emergence time or RLU or RFU. Level can be logarithmic or arithmetic. Level can be converted between different forms of expression. For example, the relationship between RFU and time can be converted to emergence time, and a calibration curve can be used to convert emergence time to a logarithmic value. As another example, a logarithmic value can be converted to an arithmetic value. In some embodiments, a calibration curve or other appropriate standards are used to help compare a level with a predetermined threshold value.

[0198] In some embodiments, the predetermined threshold value is at a value where the expected instrument error is greater than or approximately equal to the expected error due to point mutation, or at a value where the expected error caused by point mutation is greater than or approximately equal to the expected random error. Random error represents the change between repeated measurements due to error sources such as changes in biochemical processes, sample and reagent processing, and instrument performance. In some embodiments, the predetermined threshold value is within the value of about 50IU / ml, 40IU / ml, 30IU / ml, 20IU / ml, or 10IU / ml, where the expected instrument error is equal to the expected error caused by point mutation. In some embodiments, the predetermined threshold value is within about 1, 0.5, or 0.25log IU / ml of the value of the expected instrument error equal to the expected error caused by point mutation. Control experiments can be used to determine the expected error, for example, the repetition of instrument error and the comparison of point mutants with HBV nucleic acid, without the mutation of the expected error caused by point mutation.

[0199] It may be beneficial to use a predetermined threshold value around a value where the expected random error is approximately equal to the expected error due to point mutations, because when the amplicon levels are all below this value, random error is likely to be the main source of variability, and averaging may reduce the total error. On the other hand, when at least one amplicon level is above this value, mutations are more likely to be the main source of variability, so that using a higher measurement level may reduce the total error.

[0200] Exemplary predetermined threshold values ​​are provided in the above summary of the invention in IU / ml values, which are understood to be convertible to other forms as discussed above, including copies / ml, log copies / ml, emergence time, etc. In addition, alternative values ​​for the predetermined threshold values ​​may be required depending on the instrument used.

[0201] In some embodiments, the method or use can provide at least three, four, five, six, seven, eight or nine HBV genotypes (e.g., genotype A, B, C, C2, D, E, F, G or H) of substantially equivalent quantification, optionally with other non-HBV nucleic acids suspected to be present in the sample (e.g., other blood-borne pathogens) with minimal cross-reactivity. In some aspects, the method includes hepatitis A, rubella, hepatitis C, herpes simplex 1, herpes simplex 2, HIV2, parvovirus, dengue fever, CMV, HTLV, Epstein-Barr and West Nile virus. In some embodiments, the methods and uses of the present disclosure show minimal cross-reactivity to one or more or all of Candida albicans, Propionibacterium acnes, Staphylococcus aureus, Staphylococcus epidermidis or Neisseria gonorrhoeae. In one aspect, the methods and uses of the present disclosure are multiplexed with methods for detecting one or more of the aforementioned viruses or microorganisms. Generally, minimal cross-reactivity is understood to mean exhibiting at least about 95% specificity, such as at least about 96%, 97%, 98% or 99%.

[0202] Example

[0203] The following examples are provided to illustrate certain disclosed embodiments and are not to be construed as limiting the scope of the disclosure in any way.

[0204] General reagents and methods. Unless otherwise indicated, amplification is performed isothermally using transcription-mediated amplification of T7 RNA polymerase and reverse transcriptase. Dual-phase TMA is performed essentially as described in U.S. Patent No. 9,139,870, which is incorporated herein by reference. Typically, the last primer added in the dual-phase procedure is a T7 primer for each amplicon to be produced, or when a combination of two or more different T7 primer sequences is used, a shorter T7 primer for each amplicon to be produced. Exemplary amplification oligomers include those having SEQ ID NOs: 2, 3, 14, 15, 30, and 41 sequences.

[0205] Amplification reactions were performed for various primer combinations using approximately 5 to 10 pmoles of T7 primers and non-T7 primers per reaction.

[0206] Detection uses a molecular torch as a probe oligomer, which contains a 5'-fluorophore (e.g., FAM or ROX) and a 3'-quencher (e.g., DABCYL) ("5F3D" for FAM and DABCYL, or "5R3D" for ROX and DABCYL). Torches are discussed in detail in U.S. Pat. No. 6,849,412, which is incorporated by reference. Torches generally contain a -(CH2)9-linker near the 3'-end (e.g., between the 5th and 6th or between the 4th and 5th nucleotides from the 3'-end). Exemplary probe oligomers include those having the sequences of SEQ ID NOs: 29 and 40. Target capture is performed essentially as described in U.S. Pat. No. 8,034,554, which is incorporated herein by reference. Exemplary target capture oligomers include those having the sequences of SEQ ID NOs: 45-48 and 87-95.

[0207] Exemplary internal control oligomers and templates are discussed in US Pat. No. 7,785,844, which is incorporated herein by reference.

[0208] Unless otherwise indicated, "HBV Database" sequences are derived from a database of approximately 4300 HBV isolate sequences obtained from human donors.

[0209] Example 1 - Target Capture

[0210] Target capture, followed by washing, linear amplification, exponential amplification and detection, was performed using various exemplary target capture oligomers (TCOs) selected from TCOs having sequences of SEQ ID NOs: 45-48 or 87-95, both alone and in combination in the presence of an initial amplification oligomer (see Example 2). A copy to IU conversion factor of 5 copies / IU was used. In the absence of any added TCO, the sensitivity was about 4% or less when testing 2 IU / mL WHO standard HBV virus in serum, compared to a sensitivity of up to about 89% when a combination of multiple TCOs was used. Sensitivities ranging from about 30-60% were obtained using various single TCOs. This shows the benefits that can be provided by using multiple exemplary TCOs.

[0211] Example 2 - Initial Amplification of Oligos

[0212] The functionality of the T7 primary amplification oligomer was demonstrated as follows. HBV nucleic acid at concentrations of 2 IU / ml, 10 IU / ml and 2000 IU / ml was used as the starting material in the capture-amplification reaction. Target capture was performed using a combination of TCOs selected from TCOs having sequences of SEQ ID NOs: 45-48 or 87-95, followed by washing, linear amplification, exponential amplification and detection. The primary amplification oligomer was omitted in the experiment, and the results were Figure 4A -B. Therefore, it is considered that Figure 4A -B shows that no linear amplification occurred in the experiment because no amplification oligomers were added until the exponential amplification phase began. The A376 and A35 initial amplification oligomers (SEQ ID NOs: 16 and 31) were present during the capture phase of the experiment, which resulted in Figure 4C -D appears.

[0213] At 2000 IU / ml, HBV nucleic acid was detected in each experiment with a reproducible emergence time (in Figure 4A -D). At lower concentrations of HBV nucleic acid of 2 IU / ml and 10 IU / ml ( Figure 4A -D are indicated by brackets; 2 IU / ml traces are gray, 10 IU / ml traces are black), for each amplicon detected by the 408-435 probe oligomer and the 668A probe oligomer, the emergence time of the non-T7 initial amplification oligomer over a certain range of time ( Figure 4A -B) scatter was about 27 minutes higher. Using the T7 primary amplification oligo, emergence times were generally about 16-22 minutes for amplicons detected by the 408-435 probe oligo, and about 18-26 minutes for amplicons detected by the 668A probe oligo, although in isolated cases the 2 IU / ml sample showed a later emergence time. When the primary amplification oligo was used, the 2 IU / ml and 10 IU / ml emergence time data were improved because traces from these experiments showed earlier, more consistent emergence times with less overlap than when the primary amplification oligo was not present ( Figure 4C relatively Figure 4A ;and Figure 4D relatively Figure 4B ), consistent with the initial amplification oligomer contributing to the lower limit of quantitation.

[0214] Example 3-NT7 amplification oligomer (-)02

[0215] Amplification was performed on a series of HBV clone sequences corresponding to sequences from the HBV database using a set of oligomers at a concentration of 2000 IU / ml (3.3 log IU / ml), in which the NT7 (-) 02 oligomer (SEQ ID NO: 3) was lacking, and the procedure was otherwise as described in Example 2, in which two initial amplification oligomers were present. Most sequences were quantified to within about 0.25 log of the target value, but clone 4G (in which an A to G mutation was present at the NT7 binding site in the corresponding region) was underquantified by about 2 logs (not shown). Reactions with (-) 02 oligomers included at 0.2, 0.3, and 0.4 pmol / μl showed consistent quantification with other clones and within about 0.25 log of the target, and the addition of (-) 02 oligomers had minimal effect on the quantification of other clones.

[0216] Example 4 - Displacer Oligomers

[0217] HBV DNA is partially double stranded. Displacer oligonucleotides such as the 259-290 oligomer (SEQ ID NO: 67) or the 452B oligomer (SEQ ID NO: 73) can improve assay performance by generating a single stranded template through extension of the displacer oligonucleotide and concomitant strand displacement.

[0218] The effect of using a displacer oligonucleotide is shown below in Table 1. The procedure was as described in Example 2 where two initial amplification oligos were present, except that the displacer oligos used were as shown in Table 1.

[0219] Table 1. Sensitivity of different displacer oligomers for detecting HBV.

[0220] Probe N There are two types of displacers Only 259-290 Only 452B No displacer present 408-435 (SEQ ID NO: 29) 27 89％ 63％ 33％ 33％ 668A (SEQ ID NO: 40) 27 89％ 74％ 74％ 82％

[0221] Example 5 - Exemplary Internal Control Oligo

[0222] Oligos according to SEQ ID NO: 77 (NT7 amplification oligo), 78 (T7 amplification oligo), 79 (probe), and 80 (capture) were evaluated for use as internal controls (also known as generic internal controls [GIC], IC). Inclusion of appropriate IC oligos in the target capture and amplification reagents along with IC template in the target capture reagent did not significantly affect assay performance (not shown). Moderate slowdowns based on resource competition (e.g., differences in emergence time of about 1 to 3 minutes at lower target concentrations) were not considered significant effects.

[0223] Example 6-HBV subtype detection and clinical comparison

[0224] The Federal Serum and Vaccine Institute (PEI) HBV genotype reference panel and additional genotype H clinical samples (BocaBiolistics) were tested in 5 replicates. The procedure was as described in Example 2 where two initial amplification oligos were present. Figure 5 Quantitative results are shown in comparison to those obtained from commercially available Abbott and Roche assays (single replicates). The PEI group samples generally quantified within 0.3 log copies of the target, comparing favorably to the individual measurements from the Abbott and Roche assays.

[0225] Example 7 - Reproducibility, Linearity and Data Analysis

[0226] Reproducibility. Reproducibility was tested on a panel of 796 individual donor samples representing genotypes AH and a range of viral loads. The assay was as described in Example 2 including two initial amplification oligos. Quantification was reproducible over a wide range of HBV nucleic acid concentrations, such as Fig. 6A Proven in.

[0227] Linearity. Quantification was also linear with respect to the emergence time of the two amplicons detected within a range of 2 to 9 log copies / ml of double-stranded HBV DNA ( Figure 6B , amplicon detected by 408-435 probe oligomer; Figure 6C , amplicon detected by 668A probe oligomer).

[0228] Data Analysis. It was observed that the precision was lower at the low end of the concentration, for example, below about 50 IU / ml (about 1.7 log IU / ml) ( Fig. 6A It was also found that divergent measurements of Amplicon 1 and Amplicon 2 (e.g., Amplicon 1 represents a concentration below about 50 IU / ml and Amplicon 2 represents a concentration above about 50 IU / ml, or vice versa) were sometimes due to the presence of mutations in the HBV sequence that delayed amplification and thus suppressed the observed concentrations (data not shown).

[0229] In view of this phenomenon and the relatively high accuracy and precision of single amplicon quantification above about 50 IU / ml, a segmented analysis process was developed in which the assay result is reported as the maximum of the observed results of amplicon 1 and 2 if either or both of amplicon 1 and 2 are greater than or equal to 50 IU / ml, otherwise the arithmetic mean of the observed results of amplicon 1 and 2 is reported. This segmented process was found to improve overall accuracy (e.g., relative to each of (i) always reporting the mean and (ii) always reporting the maximum) (expressed as the frequency of errors exceeding a threshold such as 0.25 log copies / ml with respect to a set of targets at different genotypes and concentrations). It should be understood that with respect to overall accuracy, a shift of 50 IU / ml is not critical to obtaining a benefit; for example, shift values ​​such as about 25 IU / ml or 100 IU / ml are also expected to be effective.

[0230] Example 8 - Effect of quantification of amplicon 1+2 on accuracy and reactivity in low concentration assays

[0231] In addition to Amplicon 1, the effect of including reagents for amplification and detection of Amplicon 2 was also evaluated. The procedure was as described in Example 2, in which two initial amplification oligomers were present, except that for the data of Amplicon 1 only, the A35, A02, A08, and 668A oligomers were absent. Precision was evaluated at 10 IU / ml. Sensitivity was evaluated at 5 IU / ml.

[0232] The quantitative results of Amplicon 1+2 were as described in Example 7, using a 50 IU / ml transition value for the segmented analysis process. Including the amplification and analysis of Amplicon 2 reduced the total error for genotypes BF relative to Amplicon 1 alone (Table 2). For all genotypes, including genotypes B, D and E, the improved or maintained reactivity was about the same, with reactivity improved to 100% (Table 3).

[0233] Table 2. Accuracy of quantification of amplicon 1 relative to amplicon 1+2

[0234]

[0235] Total error = bias + 2 × (standard deviation)

[0236] Table 3. Reactivity of Amplicon 1 relative to Amplicon 1+2 detection

[0237]

[0238] Example 9 - Additional Characterization of Assay Performance

[0239] The assay described in Example 2 including two initial amplification oligomers was further characterized as follows.

[0240] Quantification Across Genotypes. Genotypes BH and C2 were both quantified within approximately 0.15 log of genotype A by either the amplicons alone or in combination as shown in Table 4. When data from two amplicons were used, each of genotypes B, C, C2, and EH were quantified within 0.1 log of genotype A, demonstrating improved consistency of quantification.

[0241] Table 4. Quantification across genotypes.

[0242]

[0243] Clinical specificity. Clinical specificity was assessed by testing 292 fresh and 747 frozen HBV-negative clinical specimens. A total of 521 plasma and 581 serum specimens were tested. Specificity was calculated as the percentage of HBV-negative samples in which HBV DNA was not detected. HBV DNA was not detected in 1038 / 1039 total samples (99.9% specificity, 95% confidence interval: 99.5-100%). A single false positive occurred in a frozen serum sample (Table 5).

[0244] Table 5. Clinical-specific results.

[0245]

[0246] Cross-reactivity. To evaluate whether the assay would cross-react with microorganisms or viruses that may be present in clinical samples, leading to false-positive results or deviations in quantitative accuracy, 5 microorganisms and 15 blood-borne viruses (BBV) were spiked into HBV-negative normal donors (Table 6). In the absence of HBV virus, none of the microorganisms caused false-positive results. In the presence of 2000 IU / mL (i.e., 10,000 copies / ml or 4 log copies / ml) of HBV virus, none of the microorganisms interfered with detection. HBV-positive samples with additional microorganisms or blood-borne viruses all showed 100% reactivity and were quantified within an average of 0.21 log copies. When detecting two amplicons, the maximum difference observed was 0.38 log copies for HIV-2 when detecting amplicon 2 and HTLV.

[0247] Table 6. Cross-reactivity analysis.

[0248]

[0249] Abbreviations: HSV, herpes simplex virus; EBV, Epstein Barr virus; HIV, human immunodeficiency virus; CMV, cytomegalovirus; HAV, hepatitis A virus; HTLV, human T-lymphotropic virus; WNV, West Nile virus.

[0250] Sequence Listing

[0251] In the table below, lower case letters indicate 2'Ome RNA and upper case letters indicate DNA.

[0252]

[0253]

[0254]

[0255]

[0256]

[0257]

[0258]

[0259]

[0260]

[0261]

[0262] Sequence Listing <110> GEN-PROBE INCORPORATED GAO, Kui LINNEN, Jeffrey M. <120> Compositions and methods for detecting or quantifying hepatitis B virus <130> DIA.0021-PCT <150> 62 / 424,956 <151> 2016-11-21 <160> 125 <170> PatentIn Version 3.5 <210> 1 <211> 3221 <212> DNA <213> Hepatitis B virus <300> <308> AB116092 <309> 2006-10-05 <313> (1)..(3221) <400> 1 ttccacagct ttccaccaag ctctgcaaga tcccagagtc aggggcctgt attttcctgc 60 tggtggctcc agttcaggaa cactcaaccc tgttccaact attgcctctc acatctcgtc 120 aatctcctcg aggattgggg accctgcacc gaacatggag aacatcacat caggattcct 180 aggacccctg ctcgtgttac aggcggggtt tttcttgttg acaagaatcc tcacaatacc 240 gcagagtcta gactcgtggt ggacttctct caattttcta gggggagcac ccgtgtgtct 300 tggccaaaat tcgcagtccc caacctccaa tcactcacca acctcctgtc ctccaatttg 360 tcctggttat cgctggatgt gtctgcggcg ttttatcata ttcctcttca tcctgctgct 420 atgcctcatc ttcttattgg ttcttctgga ttatcaaggt atgttgcccg tttgtcctct 480 aattccagga tcaacaacaa ccagcacggg accctgcaaa acctgcacga ctcctgctca 540 agggaactct atgtttccct catgttgctg tacaaaacct acggatggaa attgcacctg 600 tattcccatc ccatcatctt ggggtttcgc aaaattccta tgggagtggg cctcagtccg 660 tttctcttgt ctcagtttac tagtgccatt tgttcagtgg ttcgtagggc tttcccccac 720 tgtttggctt ttagctatat ggatgatgtg gtactggggg ccaagtctgt acaacatctt 780 gagtcccttt acaccgctgt taccaatttt cttttgtctt tgggtataca tttaaaccct 840 aacaaaacaa agagatgggg ttattccctg aatttcatgg gttatgtaat tggaagttgg 900 ggtacattgc cacaggatca tattgtacaa aaaatcaaac actgttttag aaaacttcct 960 gtaaatcgac ctattgattg gaaagtatgt cagagaattg tgggtcttttt gggctttgca 1020 gctccattta cacaatgtgg ttaccctgca ttaatgcctt tgtatgcatg tatacaggcg 1080 aaacaggctt ttactttctc gccaacttac aaggcctttc taagtaaaca gtatatgaac 1140 cttaccccg ttgcccggca acggcctggt ctgtgccaag tgtttgctga cgcaaccccc 1200 actggctggg gcttggccat cggccatcag cgcatgcgtg gaacctttgt ggctcctctg 1260 ccgatccata ctgcggaact cctagccgct tgttttgctc gcagcaggtc tggagcaaaa 1320 ctcatcggaa ctgaaattc tgtcgtcctt tctcggaagt atacatcctt tccatggctg 1380 ctaggttgta ctgccaactg gattcttcgc gggacgtcct ttgtctacgt cccgtcggcg 1440 ctgaatcccg cggacgaccc ttcgcgaggc cgcttggggc tgtatcgtcc cctctccgt 1500 ctgccgtacc gtccgaccac ggggcgcacc tctctttacg cggtctcccc gtctgtgcct 1560 tctcatctgc cggtccgtgt gcacttcgct tcacctctgc acgttgcatg gagaccaccg 1620 tgaacgccca tcaggtcctg cccaaggtct tatataagag gactcttgga ctctcagcaa 1680 tgtcaacgac cgaccttgag gcctacttca aagactgtgt gtttaaagac tgggaggagt 1740 tgggggagga gattaggtta atgatctttg tattaggagg ctgtaggcat aaattggtct 1800 gcgcaccatc atcatgcaac tttttcacct ctgcctaatc atctcttgta catgcccac 1860 ttttcaagcc tccaagctgt gccttggatg gctttggggc atggacattg acccttataa 1920 agaatttgga gctactgtgg agttactttc atttttgcct tctgacttct ttccttccgt 1980 ccgggatcta ctagatacag tcgcagctct attcgggat gccttagagt ctcctgagca 2040 ttgctcagct caccacacag cactaaggca agtcattctc tgctgggggg aattaatgac 2100 tctagctacc tgggtgggta ctaatttgca agatccagca tccagggatc tagtagtcag 2160 ttatgttaat gataacatgg gcctaaagat caggcaatta ttatggtttc atacttcttg 2220 cattacttt ggaagagaaa ctgtccttga gtatttggtc tctttcggag tgtggattcg 2280 cactcctcca gcctatagac caccaaatgc ccctatctta tcaacacttc cggaaactac 2340 tgttgttaga cgacgagacc gaggcaggtc ccctagaaga agaactccct cgcctcgcag 2400 acgaagatct caatcgccgc gtcgcagaag atctcaatct cgggaacctc aatgttagta 2460 ttccttggac tcataaggtg ggaaatttta ctggacttta ttcttctact gtccctatct 2520 ttaatcctga atggcaaaca ccgtcttttc ctaaaattca tttacatgaa gacattgcta 2580 ataggtgtca gcaatttgta ggccctctca ctgtaaatga aaaaagaaga ctgaaattaa 2640 ttatgcctgc taggttttat cctaacagca caaaatattt gcctttagac aaagggatta 2700 aaacttatta tcctgatcag gtagttaatc attactttca aacccgacat tatttacata 2760 ctctttggaa ggctgggatt ctatataaga gggaaactac acgtagcgcc tcatattgcg 2820 ggtcaccata ttcttgggaa caagagctac atcatgggag gttggtcatc aaaacctcgc 2880 aaaggcatgg ggacgaacct ttctgttccc aacccgctgg gattctttcc cgatcatcag 2940 ttggaccctg cattcggagc caatgcaaac aatccagatt gggaccctcaa ccccatcaag 3000 gaccactggc cacaagccaa ccaggtagga gtggggagcat ttggaccagg gctcactccc 3060 ccacacggag gtgttttggg gtggagccct caggctcaag gcatattggc caccgtgcca 3120 acagtgcctc ctcctgcctc caccaatcgg cagtcgggaa ggcagcctac tcccatttct 3180 ccacctctaa gagacagtca tcctcaggcc atgcagtgga a 3221 <210> 2 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 2 ggcacctagt ccagaagaac caa 23 <210> 3 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 3 ggcacctagt ccagaagaac c 21 <210> 4 <211> 10 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 4 cagaagaacc 10 <210> 5 <211> 10 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 5 tagtccagaa 10 <210> 6 <211> 10 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 6 gcacctagtc 10 <210> 7 <211> 10 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 7 gaagaaccaa 10 <210> 8 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 8 taatacgact cactatag 18 <210> 9 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 9 taatacgact cactataggg aga 23 <210> 10 <211> 31 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 10 taatacgact cactataggg agaccacaac g 31 <210> 11 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 11 aatttaatac gactcactat ag 22 <210> 12 <211> 27 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 12 aatttaatac gactcactat aggggaga 27 <210> 13 <211> 35 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 13 aatttaatac gactcactat agggagacca caacg 35 <210> 14 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 14 aatttaatac gactcactat agggagagat gtgtctgcgg cgttttatc 49 <210> 15 <211> 54 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 15 aatttaatac gactcactat agggagagat gtgtctgcgg cgttttatca tctt 54 <210> 16 <211> 57 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 16 aatttaatac gactcactat agggagagat gtgtctgcgg cgttttatca tcttcct 57 <210> 17 <211> 35 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 17 ggagagatgt gtctgcggcg ttttatcatc ttcct 35 <210> 18 <211> 27 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 18 ggagagatgt gtctgcggcg ttttatc 27 <210> 19 <211> 32 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 19 ggagagatgt gtctgcggcg ttttatcatc tt 32 <210> 20 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 20 gatgtgtctg cggcgtttta tcatcttcct 30 <210> twenty one <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> twenty one gatgtgtctg cggcgtttta tc 22 <210> twenty two <211> 27 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> twenty two gatgtgtctg cggcgtttta tcatctt 27 <210> twenty three <211> 10 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> twenty three gcgttttatc 10 <210> twenty four <211> 10 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> twenty four ctgcggcgtt 10 <210> 25 <211> 10 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 25 tgtgtctgcg 10 <210> 26 <211> 10 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 26 ttatcatctt 10 <210> 27 <211> 10 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 27 tcatcttcct 10 <210> 28 <211> 10 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 28 ggagagatgt 10 <210> 29 <211> 38 <212> RNA <213> Artificial sequence <220> <223> Synthetic oligomers <220> <221> Mixed features <222> (33)..(34) <223> Non-nucleotide C9 linker inserted between bases 33-34 <400> 29 cccacaagaa gaugaggcau agcagcagga ugaguggg 38 <210> 30 <211> 56 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 30 aatttaatac gactcactat agggagacca caacggtggg cctcagtccg tttctc 56 <210> 31 <211> 62 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <220> <221> Mixed features <222> (57)..(57) <223> n = inosine <400> 31 aatttaatac gactcactat agggagacca caacggtggg cctcagtccg tttctcntgg 60 ct 62 <210> 32 <211> 34 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 32 ggagaccaca acggtgggcc tcagtccgtt tctc 34 <210> 33 <211> 40 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <220> <221> Mixed features <222> (35)..(35) <223> n = inosine <400> 33 ggagaccaca acggtgggcc tcagtccgtt tctcntggct 40 <210> 34 <211> 29 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 34 ccacaacggt gggcctcagt ccgtttctc 29 <210> 35 <211> 35 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <220> <221> Mixed features <222> (30)..(30) <223> n = inosine <400> 35 ccacaacggt gggcctcagt ccgtttctcn tggct 35 <210> 36 <211> 10 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 36 tccgtttctc 10 <210> 37 <211> 10 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 37 ctcagtccgt 10 <210> 38 <211> 10 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 38 tgggcctcag 10 <210> 39 <211> 10 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <220> <221> Mixed features <222> (5) <223> n = inosine <400> 39 tctcntggct 10 <210> 40 <211> 26 <212> RNA <213> Artificial sequence <220> <223> Synthetic oligomers <220> <221> Mixed features <222> (21)..(22) <223> Non-nucleotide C9 linker inserted between bases 21-22 <400> 40 ugcgcuagua aacugagcca ggcgca 26 <210> 41 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 41 cctgcgaacc actgaacaaa 20 <210> 42 <211> 11 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 42 cactgaacaa a 11 <210> 43 <211> 10 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 43 gaaccactga 10 <210> 44 <211> 10 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 44 cctgcgaacc 10 <210> 45 <211> 58 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <220> <221> Mixed features <222> (26)..(58) <223> Immobilized probe-binding region: tail <400> 45 gggctttccc ccactgtttg gcttttttaa aaaaaaaaaa aaaaaaaaaa aaaaaaaa 58 <210> 46 <211> 58 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <220> <221> Mixed features <222> (26)..(58) <223> Immobilized probe-binding region: tail <400> 46 agttatatgg atgatgtggt attggtttaa aaaaaaaaaa aaaaaaaaaa aaaaaaaa 58 <210> 47 <211> 59 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <220> <221> Mixed features <222> (27)..(59) <223> Immobilized probe-binding region: tail <400> 47 ggtctgtgcc aagtgtttgc tgacgcttta aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 59 <210> 48 <211> 58 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <220> <221> Mixed features <222> (29)..(58) <223> Immobilized probe-binding region: tail <400> 48 ttgttttgct cgcagccggt ctggagcgaa aaaaaaaaaa aaaaaaaaaa aaaaaaaa 58 <210> 49 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 49 gggctttccc ccactgtttg gc 22 <210> 50 <211> 10 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 50 actgtttggc 10 <210> 51 <211> 10 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 51 cccccactgt 10 <210> 52 <211> 10 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 52 gggctttccc 10 <210> 53 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 53 agttatatgg atgatgtggt attgg 25 <210> 54 <211> 10 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 54 gtggtattgg 10 <210> 55 <211> 10 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 55 atgatgtggt 10 <210> 56 <211> 10 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 56 tatggatgat 10 <210> 57 <211> 26 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 57 ggtctgtgcc aagtgtttgc tgacgc 26 <210> 58 <211> 10 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 58 ttgctgacgc 10 <210> 59 <211> 10 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 59 agtgtttgct 10 <210> 60 <211> 10 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 60 tgccaagtgt 10 <210> 61 <211> 28 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 61 ttgttttgct cgcagccggt ctggagcg 28 <210> 62 <211> 10 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 62 gtctggagcg 10 <210> 63 <211> 10 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 63 agccggtctg 10 <210> 64 <211> 10 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 64 ctcgcagccg 10 <210> 65 <211> 33 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 65 tttaaaaaaa aaaaaaaaaa aaaaaaaaaa aaa 33 <210> 66 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 66 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 30 <210> 67 <211> 57 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 67 aatttaatac gactcactat agggagaaga ctcgtggtgg acttctctca attttct 57 <210> 68 <211> 35 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 68 ggagaagact cgtggtggac ttctctcaat tttct 35 <210> 69 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 69 agactcgtgg tggacttctc tcaattttct 30 <210> 70 <211> 10 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 70 tcaattttct 10 <210> 71 <211> 10 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 71 ttctctcaat 10 <210> 72 <211> 10 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 72 tggacttctc 10 <210> 73 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 73 tatcaaggta tgttgcccgt 20 <210> 74 <211> 10 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 74 tgttgcccgt 10 <210> 75 <211> 10 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 75 aggtatgttg 10 <210> 76 <211> 10 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 76 tatcaaggta 10 <210> 77 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 77 gattatatag gacgacaag 19 <210> 78 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 78 aatttaatac gactcactat agggagagat gattgacttg tgattccgc 49 <210> 79 <211> twenty three <212> RNA <213> Artificial sequence <220> <223> Synthetic oligomers <220> <221> Mixed features <222> (5)..(6) <223> Non-nucleotide C9 linker inserted between base positions 5-6 <400> 79 gcauggugcg aauugggaca ugc 23 <210> 80 <211> 57 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <220> <221> Mixed features <222> (25)..(57) <223> Immobilized probe-binding region: tail <400> 80 57 <210> 81 <211> twenty four <212> RNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 81 cguucacuau uggucucugc auuc 24 <210> 82 <211> 28 <212> RNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 82 aagaagauga ggcauagcag caggauga 28 <210> 83 <211> 10 <212> RNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 83 gcuaguaaac 10 <210> 84 <211> 16 <212> RNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 84 uaguaaacug agccag 16 <210> 85 <211> 20 <212> RNA <213> Artificial sequence <220> <223> Synthetic oligomers <220> <221> Mixed features <222> (18)..(19) <223> Non-nucleotide C9 linker inserted between bases 18-19 <400> 85 gcuaguaaac ugagccaggc 20 <210> 86 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 86 gatgattgac ttgtgattcc gc 22 <210> 87 <211> 59 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <220> <221> Mixed features <222> (27)..(59) <223> Immobilized probe-binding region: tail <400> 87 ggtctgtgcc aagtgtttgc tgacgcttta aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 59 <210> 88 <211> 60 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <220> <221> Mixed features <222> (31)..(60) <223> Immobilized probe-binding region: tail <400> 88 tcctctgccg atccatactg cggaactcct aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 60 <210> 89 <211> 58 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <220> <221> Mixed features <222> (29)..(58) <223> Immobilized probe-binding region: tail <400> 89 ttgttttgct cgcagccggt ctggagcgaa aaaaaaaaaa aaaaaaaaaa aaaaaaaa 58 <210> 90 <211> 56 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <220> <221> Mixed features <222> (24)..(56) <223> Immobilized probe-binding region: tail <400> 90 gggctttccc ccactgtttg gcttttaaaa aaaaaaaaaa aaaaaaaaaa aaaaaa 56 <210> 91 <211> 58 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <220> <221> Mixed features <222> (26)..(58) <223> Immobilized probe-binding region: tail <400> 91 agttatatgg atgatgtggt attggtttaa aaaaaaaaaa aaaaaaaaaa aaaaaaaa 58 <210> 92 <211> 58 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <220> <221> Mixed features <222> (26)..(58) <223> Immobilized probe-binding region: tail <400> 92 ccgctgttac caattttcttttgtctttaa aaaaaaaaaa aaaaaaaaaa aaaaaaaa 58 <210> 93 <211> 59 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <220> <221> Mixed features <222> (27)..(59) <223> Immobilized probe-binding region: tail <400> 93 59 <210> 94 <211> 58 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <220> <221> Mixed features <222> (26)..(58) <223> Immobilized probe-binding region: tail <400> 94 gucugcca aguguuugcu gacgctttaa aaaaaaaaaa aaaaaaaaaa aaaaaaaa 58 <210> 95 <211> 58 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <220> <221> Mixed features <222> (26)..(58) <223> Immobilized probe-binding region: tail <400> 95 aguuauaugg augauguggu auuggtttaa aaaaaaaaaa aaaaaaaaaa aaaaaaaa 58 <210> 96 <211> 26 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 96 ggtctgtgcc aagtgtttgc tgacgc 26 <210> 97 <211> 29 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 97 tcctctgccg atccatactg cggaactcc 29 <210> 98 <211> 28 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 98 ttgttttgct cgcagccggt ctggagcg 28 <210> 99 <211> 26 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 99 gggctttccc ccactgtttg gctttt 26 <210> 100 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 100 agttatatgg atgatgtggt attgg 25 <210> 101 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 101 ccgctgttac caattttcttttgtc 25 <210> 102 <211> 26 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 102 gguguugcc aaguguugc ugacgc 26 <210> 103 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 103 gucugcca aguguuugcu gacgc 25 <210> 104 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligomers <400> 104 aguuauaugg augaugugg auugg 25 <210> 105 <211> 960 <212> DNA <213> Hepatitis B virus <300> <308> AY707087 <309> 2007 - 10 - 23 <313> (1)..(900) <400> 105 aattccactg ccttccacca agctctgcag gatcccaaag tcaggggtct gtattttcct 60 gctggtggct ccagttcagg aacagtaaac cctgctccga atattgcctc tcacatctcg 120 tcaatctccg cgaggactgg ggaccctgtg acgaatatgg agaacatcac atcaggattc 180 ctaggacccc tgctcgtgtt acaggcgggg tttttcttgt tgacaagaat cctcacaata 240 ccgcagagtc tagactcgtg gtggacttct ctcaattttc tagggggatc acccgtgtgt 300 cttggccaaa attcgcagtc cccaacctcc aatcactcac caacctcctg tcctccaatt 360 tgtcctggtt atcgctggat gtgtctgcgg cgttttatca tattcctctt catcctgctg 420 ctatgcctca tcttcttgtt ggttcttctg gattatcaag gtatgttgcc cgtttgtcct 480 ctaattccag gaacaacaac aaccagtacg ggaccatgca aaacctgcac gactcctgct 540 caaggcaact ctatgtttcc ctcatgttgc tgtacaaaac cttcggatgg aaattgcacc 600 tgtattccca tcccatcgtc ttgggctttc gcaaaatacc tatgggagtg ggcctcagtc 660 cgtttctctt ggctcagttt actagtgcca tttgttcagt ggttcgtagg gctttccccc 720 actgtttggc tttcagctat atggatgatg tggtattggg ggccaagtct gtacagcatc 780 gtgagtccct ttataccgct gttaccaatt ttcttttgtc tctgggtata catttaaacc 840 ctaacaaaac aaaaagatgg ggttattccc taaacttcat gggttacata attggaagtt 900 ggggaacgtt gccacaggat catattgtac aaaagatcaa acactgtttt agaaaacttc 960 <210> 106 <211> 900 <212> DNA <213> Hepatitis B virus <300> <308> AB194951 <309> 2007-11-17 <313> (1)..(900) <400> 106 ttccacaaat ttccaccaag ctctgcaaga tcccagagtc aggggcctgt atcttcctgc 60 tggtggctcc agttcaggaa cagtcaaccc tgctccgaat attgcctctc acatctcgtc 120 aatctcctcg aggattgggg accctgcgcc gaacatggag aacatcacat caggattcct 180 aggacccctg ctcgtgttac aggcggggtt tttctcgttg acaaaaatcc tcacaatacc 240 gcagagtcta gactcgtggt ggacttctct caattttcta gggggagcac ccgtgtgtct 300 tggccaaaat tcgcagtccc caacctccaa tcactcacca acctcctgtc ctccaatttg 360 tcctggttat cgctggatgt gtctgcggcg ttttatcata ttcctcttca tcctgctgct 420 atgcctcatc ttcttattgg ttcttctgga ttgtcaaggt atgttgcccg tttgtcctct 480 aattccagga tccacaacca ccagtacggg accctgcaga acctgcacga ctcctgctca 540 aggcaactct atgtttccct catgttgctg tacaaaacct acggacggaa attgcacctg 600 tattcccatc ccatcatcct gggctttcgc aaaataccta tgggggtggg cctcagtccg 660 tttctcctgg ctcagtttac tagtgccatt tgttcagtgg ttcgtagggc tttcccccat 720 tgtttggctt tcagctatat ggatgatgtg gtattggggg ccaagtctgt acaacatctt 780 gagtcccttt ataccgctgt taccaatttt cttttggctt tgggcataca tttaaaccct 840 aacaaaacaa aaagatgggg ttattccctt aacttcatgg gatacataat tggaagttgg 900 <210> 107 <211> 900 <212> DNA <213> Hepatitis B virus <300> <308> AB014366 <309> 2006-11-02 <313> (1)..(900) <400> 107 ttccaccact ttccaccaaa ctcttcaaga tcccagagtc agggctctgt accttcctgc 60 tggtggctcc agttcaggaa tagtaagccc tgctcagaat actgtctctg ccatatcgtc 120 aatcttatcg acgactgggg accctgtgcc gaacatggag aacatcgcat caggactcct 180 aggacccctg ctcgtgttac aggcggggtt tttctcgttg acaaaaatcc tcacaatacc 240 acagagtcta gactcgtggt ggacttctct cagttttcta gggggaacac ccgtgtgtct 300 tggccaaaat tcgcagtccc aaatctccag tcactcacca acctgttgtc ctccaatttg 360 tcctggttat cgctggatgt atctgcggcg ttttatcatc tgcctctgca tcctgctgct 420 atgcctcatc ttcttgttgg ttcttctgga ctatcaaggt atgttgcccg tttgtcctct 480 aattccagga tcatcaacca ccagcacggg accatgcaag acctgcacaa ctcctgctca 540 aggaacctct atgtttccct catgttgctg tacaaaacct acggacggaa actgcacctg 600 tattcccatc ccatcatctt gggctttcgc aaaattccta tgggagtggg cctcagtccg 660 tttctcttgg ctcagtttac tagtgccatt tgttcagtgg ttcgtagggc tttcccccac 720 tgtctggctt tcagttatat ggatgatgtg gtattggggg ccaagtctgt acaacatctt 780 gagtcccttt atgccgctgt taccaatttt cttttgtctt tgggtataca tttaaaccct 840 cacaaaacaa aaagatgggg atattccctt aatttcatgg gatatgtaat tgggagttgg 900 <210> 108 <211> 900 <212> DNA <213> Hepatitis B virus <300> <308> AY596111 <309> 2004 - 08 - 24 <313> (1)..(900) <400> 108 ctccaccact ttccaccaaa ctcttcaaga tcccagagtc agggccctgt actttcctgc 60 tggtggctcc agttcaggaa cagtgagccc tgctcagaat actgtctctg ccatatcgtc 120 aatcttatcg aagactgggg accctgtacc gaacatggag aacatcgcat caggactcct 180 aggacccctg ctcgtgttac aggcggggtt tttctcgttg acaaaaatcc tcacaatacc 240 acagagtcta gactcgtggt ggacttctct cagttttcta ggggaaacac ccgtgtgtct 300 tggccaaaat tcgcagtccc aaatctccag tcactcacca acctgttgtc ctccaatttg 360 tcctggttat cgctggatgt gtctgcggcg ttttatcatc ttcctctgca tcctgctgct 420 atgcctcatc ttcttgttgg ttcttctgga ctatcaaggt atgttgcccg tttgtcctct 480 aattccagga tcatcaacaa ccagcaccgg accatgcaaa acctgcacaa cttctgctca 540 aggaacctct atgtttccct catgttgctg tacaaaacct acggacggaa actgcacctg 600 tattcccatc ccatcatctt gggctttcgc aaaataccta tgggagtggg cctcagtccg 660 tttctcttgg ctcagtttac tagtgccatt tgttcagtgg ttcgtagggc tttcccccac 720 tgtctggctt tcagttatat ggatgatgtg gttttggggg ccaagactgt acagcatctt 780 gagtcccttt atgccgctgt taccaatttt cttttgtctt tgggcataca tttaaaccct 840 cacaaaacaa aaagatgggg atattccctt aacttcatgg gatatgtaat tgggagttgg 900 <210> 109 <211> 900 <212> DNA <213> Hepatitis B virus <300> <308> M54923 <309> 2000-06-13 <313> (1)..(900) <400> 109 ctccaccacg ttccaccaaa ctcttcaaga tcccagagtc agggctctgt actttcctgc 60 tggtggctcc agttcaggaa cagtaaaccc tgttcagaac actgtctctt ccatatcgtc 120 aatcttatcg aagactgggg accctgtgcc gaacatggag aacatcgcat caggactcct 180 aggacccctg ctcgtgttac aggcggggtt tttcttgttg acaaaaatcc tcacaatacc 240 acagagtcta gactcgtggt ggacttctct caattttcta gggggaacac ccgtgtgtct 300 tggccaaaat tcgcagtccc aaatctccag tcactcacca acttgttgtc ctccgatttg 360 tcctggttat cgctggatgt gtctgcggcg ttttatcatc ttcctctgca tcctgctgct 420 atgcctcatc ttcttgttgg ttcttctgga ctatcaaggt atgttgcccg tttgtcctct 480 aattccagga tcatcaacca ccagcaccgg accatgcaaa acctgcacga ctcctgctca 540 aggaacctct atgtttccct catgttgctg tacaaaacct acggacggaa actgcacctg 600 tattcccatc ccatcatctt gggctttcgc aaaataccta tgggagtggg cctcagtccg 660 tttctcttgg ctcagtttac tagtgccatt tgttcagtgg ttcgtagggc tttcccccac 720 tgtctggctt tcagttatat ggatgatgtg gttttggggg ccaagtctgt acaacatctt 780 gagtcccttt atgccgctgt taccaatttt cttttgtctt tgggtataca tttaaaccct 840 cagaaaacaa aaagatgggg ctactccctc aacttcatgg ggtatgtaat tggaagttgg 900 <210> 110 <211> 900 <212> DNA <213> Hepatitis B virus <300> <308> AB073835 <309> 2002 - 06 - 04 <313> (1)..(900) <400> 110 ctccaccact ttccatcaaa ctcttcaaga tcccagagtc agggctctgt actttcctgc 60 tggtggctcc agttcaggaa tagtaaaccc tgctcagaat actgcctctg ccatatcatc 120 aaccttctcg aagactgggg accctgtacc gaacatggag aacatcgcat caggactcct 180 aggacccctg ctcgtgttac aggcggggtt tttctcgttg acaaaaatcc tcacaatacc 240 acagagtcta gactcgtggt ggacttctct caattttcta gggggaacac ccgtgtgtct 300 tggccaaaat tcgcagtccc aaatctccag tcactcacca acttgttgtc ctccaacttg 360 tcctggttat cgctggatgt atctgcggcg ttttatcata ttcctctgca tcctgctgct 420 atgcctcatc ttcttgttgg ttcttctgga ctatcaaggt atgttgcccg tttgtcctct 480 aattccagga tcatcaacca ccagcaccgg accctgcaga acctgcacga ctcctgctca 540 aggaacctct atgtttccct catgttgctg tacaaaacct acggacggaa actgcacctg 600 tattcccatc ccatcatctt gggctttcgc aaaattccta tgggagtggg cctcagtccg 660 tttctcttgg ctcagtttac tagtgccgtt tgttcagtgg ttcgtagggc tttcccccac 720 tgtctggctt tcagttatat ggatgatgtg gtattggggg ccaaatctgt acaacatctt 780 gagtcccttt atgccgctgt taccaatttt cttttgtctt tgggtataca tttaaaccct 840 cacaaaacaa aaagatgggg atattccctt aatttcatgg gatatgtaat tggtagttgg 900 <210> 111 <211> 900 <212> DNA <213> Hepatitis B virus <300> <308> AY123424 <309> 2002 - 08 - 16 <313> (1)..(900) <400> 111 aactccacaa cattccacca agctctgcta gaccccagag tgaggggcct atactttcct 60 gctggtggct ccagttccgg aacagtaaac cctgttccga ctactgcctc acccatatcg 120 tcaatcttct cgaggactgg ggaccctgca ccgaacatgg agagcacaac atcaggattc 180 ctaggacccc tgctcgtgtt acaggcgggg tttttcttgt tgacaagaat cctcacaata 240 ccacagagtc tagactcgtg gtggacttct ctcaattttc tagggggagc acccacgtgt 300 cttggccaaa attcgcagtc cccaacctcc aatcactcac caacctcttg tcctccaatt 360 tgtcctggtt atcgttggat gtgtctgcgg cgttttatca tattcctctt catcctgctg 420 ctatgcctca tcttcttgtt ggttcttctg gactaccaag gtatgttgcc cgtttgtcct 480 ctacttccaa gaacatcaac taccagcacg ggaccatgca agacctgcac gattcctgct 540 caaggaacct ctatgtttcc ctcttgttgc tgtacaaaac cttcggacgg aaactgcact 600 tgtattccca tcccatcatc ttgggctttc gcaagattcc tatgggagtg ggcctcagtc 660 cgtttctcct ggctcagttt actagtgcca tttgttcagt ggttcgtagg gctttccccc 720 actgtttggc tttcagttat atggatgatg tggtattggg ggccaagtct gtacaacatc 780 ttgagtccct ttttacctct attaccaatt ttcttttgtc tttgggtata catttgaacc 840 ccaataaaac caaacgttgg ggctattccc ttaatttcat gggatatgta attggatgtt 900 <210> 112 <211> 900 <212> DNA <213> Hepatitis B virus <300> <308> AF223955 <309> 2001 - 09 - 07 <313> (1)..(900) <400> 112 ctccagcaca ttccaccaag ctctgctaga tcccagagtg aggggcctat actttcctgc 60 tggtggctcc agttccggaa cagtaaaccc tgttccgact actgcctctc ccatatcgtc 120 aatcttctcg aggactgggg accctgcacc gaatatggag agcaccacat caggattcct 180 aggacccctg ctcgtgttac aggcggggtt tttcttgttg acaagaatcc tcacaatacc 240 acagagtcta gactcgtggt ggacttctct caattttcta gggggagcac ccacgtgtcc 300 tggccaaaat ttgcagtccc caacctccaa tcactcacca acctcttgtc ctccaatttg 360 tcctggttat cgctggatgt gtctgcggcg ttttatcatc ttcctcttca tcctgctgct 420 atgcctcatc ttcttgttgg ttcttctgga ctaccaaggt atgttgcccg tttgtcctct 480 acttccagga acatcaacta ccagcacggg accatgcagg acctgcacga ttcctgctca 540 aggaacctct atgtttccct cttgttgctg tacaaaacct tcggacggaa attgcacttg 600 tattcccatc ccatcatctt gggctttcgc aagattccta tgggagtggg cctcagtccg 660 tttctcctgg ctcagtttac tagtgccatt tgttcagtgg ttcgtagggc tttcccccac 720 tgtttggctt tcagttatat ggatgatgtg gtattggggg ccaagtctgt acaacatctt 780 gaatcccttt ttaccgctat taccaatttt cttttgtctt tgggtataca tttaaaccct 840 aataaaacca aacgttgggg ctactccctt aactttatgg gatatgtcat tggaagttgg 900 <210> 113 <211> 900 <212> DNA <213> Hepatitis B virus <300> <308> X75665 <309> 2006 - 11 - 14 <313> (1)..(900) <400> 113 ctccacaaca ttccaacaag ctctgcagga tcccagagtc agggtccttt attttcctgc 60 tggtggctcc agttccggaa cagtaaaccc tgttccgact actgcctctc tcatttcgtc 120 aatcttctcg aggattgggg accctgtaac gaacatggag aacacaacat caggattcct 180 aggacccctg ctcgtgttac aggcggggtt tttcttgttg acaaaaatcc tcacaatacc 240 acagagtcta gactcgtggt ggacttctct caattttcta gggggagcac ccgtgtgtcc 300 tggccaaaat tcgcagtccc caacctccaa tcactcacca acctcttgtc ctccaatttg 360 tcctggctat cgctggatgt gtctgcggcg ttttatcatc ttcctcttca tcctgctgct 420 atgcctcatc ttcttgttgg ttcttctgga ctaccaaggt atgttgcccg tttgtcctct 480 acttccagga acatcaacta ccagcacggg accatgcaag acctgcacga ttcctgctca 540 aggaacctct atgtttccct catgttgctg tacaaaacct tcggacggaa actgcacttg 600 tattcccatc ccatcatcct gggctttcgt aagattccta tgggagtggg cctcagtccg 660 tttctcctgg ctcagtttac tagcgccatt tgttcagtgg ttcgtagggc tttcccccac 720 tgtttggctt tcagttatat ggatgatgtg gtattggggg ccaagtctgt acaacatctt 780 gagtcccttt atacctctat taccaatttt cttttgtctt tgggtataca tttaaaccct 840 aataaaacca aaagatgggg ctattccctt aacttcatgg gctatgtaat tggaagttgg 900 <210> 114 <211> 900 <212> DNA <213> Hepatitis B virus <300> <308> AB104712 <309> 2003 - 06 - 26 <313> (1)..(900) <400> 114 aactccacta ccttccacca aactctgcaa gatcccagag tgagaggcct gtatttccct 60 gctggtggct ccagttcagg aacagtaaac cctgttccga ctactgtctc tcacatatcg 120 tcaatcttct cgaggattgg ggaccctgcg ctgaacatgg agaacatcac atcaggattc 180 ctaggacccc tgctcgtgtt acaggcgggg tttttcttgt tgacaagaat cctcacaata 240 ccgcagagtc tagactcgtg gtggacttct ctcaattttc taggggaac taccgtgtgt 300 cttggccaaa attcgcagtc cccaacctcc aatcactcac caacctcctg tcctccaact 360 tgtcctggtt atcgctggat gtgtctgcgg cgttttatca tcttctctt catcctgctg 420 ctatgcctca tcttcttgtt ggttcttctg gactatcaag gtatgttgcc cgtttgtcct 480 ctaattccag gatcttcaac taccagcacg ggaccatgca gaacctgcac gactcctgct 540 caaggaacct ctatgtatcc ctcctgctgc tgtaccaaac cttcggacgg aaattgcacc 600 tgtattccca tcccatcatc ctgggctttc ggaaaattcc tatgggagtg ggcctcagcc 660 cgtttctcct ggctcagttt actagtgcca tttgttcagt ggttcgtagg gctttcccc 720 actgtttggc tttcagttat atggatgatg tggtattggg ggccaagtct gtacagcatc 780 ttgagtccct ttttaccgct gttaccaatt ttcttttgtc tttgggtata catttaaacc 840 ctaacaaaac aaaaagatgg ggttactctt tacatttcat gggttatgtc attggatgtt 900 <210> 115 <211> 900 <212> DNA <213> Hepatitis B virus <300> <308> AB205126 <309> 2008-11-14 <313> (1)..(900) <400> 115 aactccacta ccttccacca aactctgcaa gatcccagag tgagaggcct gtatttccct 60 gctggtggct ccagttcagg aacagtaaac cctgttccga ctactgtctc tcacatatcg 120 tcaatcttct cgaggattgg ggaccctgcg ctgaacatgg agaacatcac atcaggattc 180 ctaggacccc tgctcgtgtt acaggcgggg tttttcttgt tgacaagaat cctcacaata 240 ccgcagagtc tagactcgtg gtggacttct ctcaattttc tagggggaac taccgtgtgt 300 cttggccaaa attcgcagtc cccaacctcc aatcactcac caacctcctg tcctccaact 360 tgtcctggtt atcgctggat gtgtctgcgg cgttttatca tcttcctctt catcctgctg 420 ctatgcctca tcttcttgtt ggttcttctg gactatcaag gtatgttgcc cgtttgtcct 480 ctaattccag gatcttcaac taccagcacg ggaccatgca gaacctgcac gactcctgct 540 caaggaacct ctatgtatcc ctcctgctgc tgtaccaaac cttcggacgg aaattgcacc 600 tgtattccca tcccatcatc ctgggctttc ggaaaattcc tatgggagtg ggcctcagcc 660 cgtttctcct ggctcagttt actagtgcca tttgttcagt ggttcgtagg gctttccccc 720 actgtttggc tttcagttat atggatgatg tggtattggg ggccaagtct gtacagcatc 780 ttgagtccct ttttaccgct gttaccaatt ttcttttgtc tttgggtata catttaaacc 840 ctaacaaaac aaaaagatgg ggttactctt tacatttcat gggttatgtc attggatgtt 900 <210> 116 <211> 900 <212> DNA <213> Hepatitis B virus <300> <308> AY233291 <309> 2004 - 04 - 23 <313> (1)..(900) <400> 116 ttccacaacc ttccaccaaa ctctgcaaga tcccagagtg agaggcctgt atttccctgc 60 tggtggctcc agttcaggaa cagtaaaccc tgttccgact actgcctctc ccttatcgtc 120 aatcttctcg aggattgggg accctgcgcc gaacatggag aacatcacat caggattcct 180 aggacccctg ctcgtgttac aggcggggtt tttcttgttg acaagaatcc tcacaatacc 240 gcagagtcta gactcgtggt ggacttctct caattttcta gggggaacta ccgtgtgtct 300 tggccaaaat tcgcagtccc caacctccaa tcactcacca acctcctgtc ctccaacttg 360 tcctggttat cgctggatgt gtctgcggcg ttttatcatc ttcctcttca tcctgctgct 420 atgcctcatc ttcttgttgg ttcttctgga ctatcaaggt atgttgcccg tgtgtcctct 480 aattccagga tcctcaacca ccagcacggg accatgccga acctgcacga ctcctgctca 540 aggaacctct atgtatccct cctgttgctg taccaaacct tcggacggaa attgcacctg 600 tattcccatc ccatcatcct ggcctttcgg aaaattccta tgggagtggg cctcagcccg 660 tttctcctgg ctcagtttac tagtgccatt tgttcagtgg ttcgtagggc tttcccccac 720 tgtttggctt tcagttatat ggatgatgtg gtattggggg ccaagtctgt acagcatctt 780 gagtcccttt ttaccgctgt taccaatttt cttttgtctt tgggtataca tttaaaccct 840 aacaaaacaa agagatgggg ttactctcta aattttatgg ggtatgtcat tggatgttat 900 <210> 117 <211> 900 <212> DNA <213> Hepatitis B virus <300> <308> AB048702 <309> 2001-03-23 <313> (1)..(900) <400> 117 ctccacaacc ttccaccaaa ctctgcaaga tcccagagtg agaggcctgt atctccctgc 60 tggtggctcc agttcaggaa cagtaaaccc tgttccgact actgcctctc ccatatcgtc 120 aatcttctcg aggattgggg accttgcgct gaacatggag aacatcacat caggattcct 180 aggacccctg ctcgtgttac aggcggggtt tttcttgttg acaagaatcc tcacaatacc 240 gcagagtcta gactcgtggt ggacttctct caattttcta gggggaacta ccgtgtgtct 300 tggccaaaat tcgcagtccc caacctccaa tcactcacca acctcctgtc ctccaacttg 360 tcctggttat cgctggatgt ttctgcggcg ttttatcatc ttcctcttca tcctgctgct 420 atgcctcatc ttcttgttgg ttcttctgga ttatcaaggt atgttgcccg tttgtcctct 480 aattccagga tcatcaacca ccagcacggg accctgcaga acctgcacga ctcctgctca 540 aggaacctct atgtatccct cctgttgctg tacaaaacct tcggatggaa actgcacctg 600 tattcccatc ccatcatcct gggctttcgg aaaattccta tgggagtggg cctcagcccg 660 tttctcttgg ctcagtttac tagtgccatt tgttcagtgg ttcgtagggc tttcccccac 720 tgtttggctt tcagttatat ggatgatgtg gtattggggg ccaagtctgt acagcacctt 780 gagtcccttt ttaccgctgt taccaatttt cttttgtctt tgggtataca tttaaaccct 840 aacaaaacaa aaagatgggg ttattctcta aatttcatgg gctatgtcat tggaagttgg 900 <210> 118 <211> 900 <212> DNA <213> Hepatitis B virus <300> <308> AB205191 <309> 2006 - 01 - 12 <313> (1)..(900) <400> 118 ttccacaaca ttccaccaag ctctgcagga tcccagagta agaggcctgt attttcctgc 60 tggtggctcc agttccggaa cagtgaaccc tgttccgact actgcctcac tcatctcgtc 120 aatcttctcg aggattgggg accctgcacc gaacatggaa ggcatcacat caggattcct 180 aggacccctg ctcgtgttac aggcggggtt tttcttgttg acaaaaatcc tcacaatacc 240 gcagagtcta gactcgtggt ggacttctct caattttcta gggggagctc ccgtgtgtct 300 tggccaaaat tcgcagtccc caacctccaa tcactcacca acctcttgtc ctccaatttg 360 tcctggctat cgctggatgt gtctgcggcg ttttatcatc ttcctcttca tcctgctgct 420 atgcctcatc ttcttgttgg ttcttctgga ctatcaaggt atgttgcccg tttgtcctct 480 aattccagga tcatcaacca ccagtacggg accctgccga acctgcacga ctcttgctca 540 aggaacctct atgtttccct catgttgctg ttcaaaacct tcggacggaa attgcacttg 600 tattcccatc ccatcatcat gggctttcgg aaaattccta tgggagtggg cctcagcccg 660 tttctcctgg ctcagtttac tagtgccatt tgttcagtgg ttcgccgggc tttcccccac 720 tgtctggctt tcagttatat ggatgatgtg gtattggggg ccaagtctgt acaacatctt 780 gagtcccttt ataccgctgt taccaatttt cttttgtctt tgggtataca tttaaatcca 840 aacaaaacaa aaagatgggg atattcccta aatttcatgg gttatgtaat tggaagttgg 900 <210> 119 <211> 900 <212> DNA <213> Hepatitis B virus <300> <308> AY090459 <309> 2002 - 08 - 07 <313> (1)..(900) <400> 119 gaactcaact cagttccacc aggctctgtt agatccgagg gtaagggctc tgtattttcc 60 tgctggtggc tccagttcag agacacagaa ccctgctccg actattgcct ctctcacatc 120 atcaatcttc ttgaagactg ggggccctgc tatgaacatg gacaacatca catcaggact 180 cctaggaccc ctgctcgtgt tacaggcggt gtgtttcttg ttgacaaaaa tcctcacaat 240 accacagagt ctagactcgt ggtggacttc tctcaatttt ctagggggaa caccagggtg 300 tcctggccaa aattcgcagt ccccaacctc caatcactta ccaacctcct gtcctccaac 360 ttgtcctggt tatcgctgga tgtgtctgcg gcgttttatc atcttcctct tcatcctgct 420 gctatgcctc atcttcttgt tggttcttct ggactatcaa ggtatgttgc ccgtttgtcc 480 tctacttcca ggatccacga ccaccagcac gggaccatgc aaaacctgca caactcttgc 540 tcaaggaacc tctatgtttc cctcctgctg ctgttccaaa ccttcggacg gaaactgcac 600 ttgtattccc atcccatcat cctgggcttt aggaaaatac ctatgggagt gggcctcagc 660 ccgtttctcc tggctcagtt tactagtgca atttgttcag tggtgcgtag ggctttcccc 720 cactgtttgg cttttagtta tatggatgat ctggtattgg gggccaaatc tgtgcagcat 780 cttgagtccc tttataccgc tgttaccaat tttctgttat ctgtgggtat ccatttaaat 840 acctctaaaa cgaaaagatg gggctatact ttaaatttca tgggatatgt tattggcagt 900 <210> 120 <211> 900 <212> DNA <213> Hepatitis B virus <300> <308> AF223963 <309> 2001-01-17 <313> (1)..(900) <400> 120 ctccactcag ttccaccagg ctctgttaga tccgagggta agggctctgt attttcctgc 60 tggtggctcc agttcagaga cacagaaccc tgctccgact attgcctctc tcacatcatc 120 aatcttcttg aagactgggg gccctgctac gaacatggac aacatcacat caggactcct 180 aggacccctg ctcgtgttac aggcggtgtg tttcttgttg acaaaaatcc tcacaatacc 240 acagagtcta gactcgtggt ggacttctct caattttcta gggggaacac ccgggtgtcc 300 tggccaaaat tcgcagtccc caacctccaa tcacttacca acctcctgtc ctccaacttg 360 tcctggctat cgctggatgt gtctgcggcg ttttatcatc ttcctcttca tcctgctgct 420 atgcctcatc ttcttgttgg ttcttgtgga ctatcaaggt atgttgcccg tttgtcctcc 480 acttccagga tccacgacca ccagcacggg accatgcaaa acctgcacaa ctcttgctca 540 aggaacctct atgtttccct cttgctgctg ttccaaaccc tcggacggaa actgcacttg 600 tattcccatc ccatcatcct gggctttagg aaaataccta tgggagtggg cctcagcccg 660 tttctcctgg ctcagtttac tagtgcaatt tgttcagtgg tgcgtagggc tttcccccac 720 tgtctggctt ttagttatat ggatgatctg gtattggggg ccaaatctgt gcagcatctt 780 gagtcccttt ataccgctgt taccaatttt ctgttatctg tgggtatcca tttaaatacc 840 tcgaaaacaa aaagatgggg ttatacccta aatttcatgg gttatgttat tggcagttgg 900 <210> 121 <211> 900 <212> DNA <213> Hepatitis B virus <300> <308> AY311369 <309> 2006 - 08 - 28 <313> (1)..(900) <400> 121 ctcaacccag ttccaccagg ctctgttgga tcccagggta agggctctgt acttccctgc 60 tggtggctcc agttcaggga cacagaaccc tgctccgact attgcctctc tcacatcatc 120 aatcttctcg aagactgggg gccctgctat gaacatggac aacattacat caggactcct 180 aggacccctg ctcgtgttac aggcggtgtg tttcttgttg acaaaaatcc tcacaatacc 240 acagagtcta gactcgtggt ggacttctct caattttcta gggggactac ccgggtgtcc 300 tggccaaaat tcgcagtccc caacctccaa tcacttacca acctcctgtc ctccaacttg 360 tcctggctat cgttggatgt gtctgcggcg ttttatcatc ttcctcttca tcctgctgct 420 atgcctcatc ttcttgttgg ttcttctgga ctatcaaggt atgttgcccg tttgtcctct 480 acttccagga tccacgacca ccagcacggg accctgcaaa acctgcacaa ctcttgcaca 540 aggaacctct atgtttccct cctgttgctg ttccaaaccc tcggacggaa actgcacttg 600 tattcccatc ccatcatctt gggctttagg aaaataccta tgggagtggg cctcagcccg 660 tttctcctgg ctcagtttac tagtgcaatt tgttcagtgg tgcgtcgggc tttcccccac 720 tgtttggctt ttagttatat ggatgatctg gtattggggg ccaaatctgt gcagcatctt 780 gagtcccttt ataccgctgt taccaatttt ctgttatctg tgggtatcca tttaaatacc 840 tctaaaacaa aaagatgggg gtactcccta cattttatgg gctatgtcat tggtagttgg 900 <210> 122 <211> 900 <212> DNA <213> Hepatitis B virus <300> <308> AB036915 <309> 2001 - 02 - 20 <313> (1)..(900) <400> 122 ctcaacccag ttccaccagg ctctgttaga tccgagggta agggctctgt attttcctgc 60 tggtggctcc agttcaggga cacagaaccc tgttccgact attgcctctc tcacatcatc 120 aatcttctcg aagactgggg gccctgctat gaacatggag aacatcacat caggactcct 180 aggacccctg ctcgtgttac aggcggtgtg tttcttgttg acaaaaatcc tcacaatacc 240 acagagtcta gactcgtggt ggacttctct caattttcta gggggactac ccgggtgtcc 300 tggccaaaat tcgcagtccc caacctccaa tcacttacca acctcctgtc ctccaacttg 360 tcctggctat cgttggatgt gtctgcggcg ttttatcatc ttcctcttca tcctgctgct 420 atgcctcatc ttcttgttgg ttcttctgga ctatcaaggt atgttgcccg tttgtcctct 480 acttccagga tccacaacca ccagcacggg accatgcaaa acctgcacaa ctcttgctca 540 aggaacctct atgtttccct cctgttgctg ttccaaaccc tcggacggaa actgcacctg 600 tattcccatc ccatcatctt gggctttagg aaaataccta tgggagtggg cctcagcccg 660 tttctcctgg ctcagtttac tagtgcaatt tgttcagtgg tgcgtagggc tttcccccac 720 tgtctggctt ttagttatat ggatgatctg gtattggggg ccaaatctgt gcagcatctt 780 gagtcccttt ataccgctgt taccaatttt ttgttatctg tgggtatcca tttaaatact 840 tctaaaacaa aaagatgggg ttacaaccta catttcatgg gttatgttat tggtagttgg 900 <210> 123 <211> 900 <212> DNA <213> Hepatitis B virus <300> <308> AB166850 <309> 2010 - 02 - 09 <313> (1)..(900) <400> 123 ctcaacccag ttccaccagg ccctgttgga tccgagggta agggctctgt ctcctcctgc 60 tggtggctcc agttcagaga cacagaaccc tgctccgact attgcctctc tcacatcatc 120 aatcttctcg aaaactgggg gccctgctat gaacatggac aacatcacat caggactcct 180 aggacccctg ctcgtgttac aggcggtgtg tttcttgttg acaaaaatcc gcacaatacc 240 acagagtcta gacttgtggt ggacttctct caattttcta gggggactac ccgggtgtcc 300 tggccaaaat tcgcagtccc caacctccaa tcacttacca acctcctgtc ctccaacttg 360 tcctggctat cgttggatgt gtctgcggcg ttttatcatc ttcctcttca tcctgctgct 420 atgcctcatc ttcttgttgg ttcttctgga ctatcaaggt atgttgcccg tttgtcctct 480 aattccagga tctacgacca ccagcacggg accatgcaaa acctgcacaa ctcttgctca 540 aggaacctct atgtttccct cctgttgctg ttcaaaacct tcggacggaa actgcacctg 600 tattcccatc ccatcatctt gggctttagg aaaataccta tgggagtggg cctcagcccg 660 tttctcctgg ctcagtttac tagtgcaatt tgttcagtgg tgcgtagggc tttcccccac 720 tgtctggctt ttagttatat ggatgatctg gtattggggg ccaaatctgt gcagcatctt 780 gagtcccttt ataccgctgt taccaatttt ctgttatctg tgggtatcca tttgaatacc 840 tctaaaacaa aaagatgggg ttacaattta catttcatgg gttatatcat tggtagttgg 900 <210> 124 <211> 900 <212> DNA <213> Hepatitis B virus <300> <308> AB064312 <309> 2002 - 06 - 04 <313> (1)..(900) <400> 124 ctctacagca ttccaccaag ctctacaaaa tcccacagtc aggggcctgt atcttcctgc 60 tggtggctcc agttcaggga tagtgaaccc tgttccgact attgcctctc acatctcgtc 120 aatcttctcc aggattgggg accctgcacc gaacatggag aacatcacat caggattcct 180 aggacccctg ctcgtgttac aggcggggtt tttcttgttg acaagaatcc tcacaatacc 240 gcagagtcta gactcgtggt ggacttctct caattttcta gggggagtgc ccgtgtgtcc 300 tggcctaaat tcgcagtccc caacctccaa tcactcacca atctcctgtc ctccaacttg 360 tcctggctat cgctggatgt gtctgcggcg ttttatcata ttcctcttca tcctgctgct 420 atgcctcatc ttcttgttgg ttcttctgga ctatcaaggt atgttgcccg tttgtcctct 480 gattccagga tcctcgacca ccagtacggg accctgcaaa acctgcacga ctcctgctca 540 aggcaactct atgtatccct catgttgctg tacaaaacct tcggacggaa attgcacctg 600 tattcccatc ccatcatctt gggctttcgc aaaataccta tgggagtggg cctcagtccg 660 tttctcttgg ctcagtttac tagtgccatt tgttcagtgg ttcgtagggc tttcccccac 720 tgtctggctt tcagctatat ggatgatgtg gtattggggg ccaaatctgt acaacatctt 780 gagtcccttt ataccgctgt taccaatttt cttttgtctt tgggtataca tctaaaccct 840 gacaaaacaa aaagatgggg ttattcctta aattttatgg gatatgtaat tggaagttgg 900 <210> 125 <211> 900 <212> DNA <213> Hepatitis B virus <300> <308> AB059659 <309> 2002 - 12 - 04 <313> (1)..(900) <400> 125 ctcaacacag ttccaccaag cactgttaga tccgagagta aggggtctgt attttcctgc 60 tggtggctcc agttcagaaa cacagaaccc tgctccgact attgcctctc tcacatcatc 120 aatcttctcg aagactgggg accctgctat gaacatggag aacatcacat caggactcct 180 aggacccctt cccgtgttac agggggtgtt tttctcgttg acaaaaatcc tcacaatacc 240 acagagtcta gactcgtggt ggacttctct caattttcta gaggtaccac ccgggtgtcc 300 tggccaaaat tcgcagtccc caatctccaa tcacttacca acctcctgtc ctccaacttg 360 tcctggctat cgttggatgt gtctgcggcg ttttatcatc ttcctcttca tcctgctgct 420 atgcctcatc ttcttgttgg ttcttctgga ctatcaaggt atgttgcccg tgtgtcctct 480 acttccagga tctacaacca ccagcacggg accctgcaaa acctgcacca ctcttgctca 540 aggaacctct atgtttccct cctgctgctg taccaaacct tcggacggaa attgcacctg 600 tattcccatc ccatcatctt gggctttcgg aaaataccta tgggagtggg cctcagcccg 660 tttctcttgg ctcagtttac tagtgcaatt tgttcagtgg tgcgtagggc tttcccccac 720 tgtctggctt ttagttatat ggatgatttg gtattggggg ccaaatctgt gcagcatctt 780 gagtcccttt ataccgctgt taccaatttt ttgttatctg tgggcatcca tttgaacaca 840 gctaaaacaa aaaggtgggg ttattcctta cactttatgg gttatataat tgggagttgg 900

Claims

1. A composition or kit comprising at least a first, a second, a third and a fourth amplification oligomer, wherein: The target hybridization sequence of the first amplification oligomer is SEQ ID NO: 2; The target hybridization sequence of the second amplification oligomer is one of SEQ ID NO: 20, 21 or 22; The target hybridization sequence of the third amplification oligomer is SEQ ID NO: 41; and The target hybridization sequence of the fourth amplification oligomer is one of SEQ ID NO: 34 or 35; Wherein the composition or kit further comprises at least one probe oligomer, wherein: a) the probe oligomer is configured to specifically hybridize to the amplicon produced by the first and the second amplification oligomers, and wherein the sequence of the probe oligomer is SEQ ID NO: 29; or b) the probe oligomer is configured to specifically hybridize to the amplicons produced by the third and fourth amplification oligomers, and wherein the sequence of the probe oligomer is SEQ ID NO:

40.

2. A composition or kit comprising at least a first, a second, a third and a fourth amplification oligomer, wherein: The target hybridization sequence of the first amplification oligomer is one of SEQ ID NO: 2 or 3; The target hybridization sequence of the second amplification oligomer is one of SEQ ID NO: 20, 21 or 22; The target hybridization sequence of the third amplification oligomer is SEQ ID NO: 41; The target hybridization sequence of the fourth amplification oligomer is SEQ ID NO: 35; Wherein the composition or kit further comprises at least one probe oligomer, wherein: a) the probe oligomer is configured to specifically hybridize to the amplicon produced by the first and second amplification oligomers, wherein the sequence of the probe oligomer is SEQ ID NO: 29; or b) the probe oligomer is configured to specifically hybridize to the amplicons produced by the third and fourth amplification oligomers, wherein the sequence of the probe oligomer is SEQ ID NO:

40.

3. A composition or kit comprising at least a first, a second, a third and a fourth amplification oligomer, wherein: The target hybridization sequence of the first amplification oligomer is one of SEQ ID NO: 2 or 3; The target hybridization sequence of the second amplification oligomer is one of SEQ ID NO: 20, 21 or 22; The target hybridization sequence of the third amplification oligomer is SEQ ID NO: 41; The target hybridization sequence of the fourth amplification oligomer is one of SEQ ID NO: 34 or 35; Wherein the composition or kit further comprises at least one probe oligomer, wherein: a) the probe oligomer is configured to specifically hybridize to the amplicon produced by the first and the second amplification oligomers, and wherein the sequence of the probe oligomer is SEQ ID NO: 29; or b) the probe oligomer is configured to specifically hybridize to the amplicons produced by the third and fourth amplification oligomers, and wherein the sequence of the probe oligomer is SEQ ID NO:

40.

4. Use of a composition for preparing a medicament for detecting hepatitis B virus in a sample, wherein the composition comprises at least a first, a second, a third and a fourth amplification oligomer, in: The target hybridization sequence of the first amplification oligomer is one of SEQ ID NO: 2 or 3; The target hybridization sequence of the second amplification oligomer is one of SEQ ID NO: 20, 21 or 22; The target hybridization sequence of the third amplification oligomer is SEQ ID NO: 41; and The target hybridization sequence of the fourth amplification oligomer is one of SEQ ID NO: 34 or 35; wherein a nucleic acid amplification reaction performed in the composition in the presence of hepatitis B virus nucleic acid produces at least a first and a second amplicons that are quantified, and wherein the first amplicon is produced by extension of the first and second amplification oligomers in the presence of the hepatitis B virus nucleic acid; and The second amplicon is produced by extension of the third and fourth amplification oligomers in the presence of the hepatitis B virus nucleic acid; and Wherein the composition further comprises at least one probe oligomer, wherein: a) the probe oligomer is configured to specifically hybridize to the amplicon produced by the first and the second amplification oligomers, and wherein the sequence of the probe oligomer is SEQ ID NO: 29; or b) the probe oligomer is configured to specifically hybridize to the amplicons produced by the third and fourth amplification oligomers, and wherein the sequence of the probe oligomer is SEQ ID NO:

40.

5. The use according to claim 4, wherein the nucleic acid amplification reaction is isothermal.

6. The use according to claim 4 or 5, wherein the nucleic acid amplification reaction is transcription-mediated amplification.

7. The use according to any one of claims 4 to 6, further comprising: Linear amplification prior to a nucleic acid amplification reaction in the presence of hepatitis B virus nucleic acid to produce at least first and second amplicons, wherein the nucleic acid amplification reaction is performed by extending at least one amplification oligomer.

8. The use according to any one of claims 4 to 7, wherein the nucleic acid amplification reaction carried out in the presence of hepatitis B virus nucleic acid that produces at least the first and second amplicons is exponential amplification.

9. The composition or kit of any one of claims 1-3 or the use of any one of claims 4-8, wherein one or more of the first, second, third and fourth amplification oligomers are promoter-primers, and / or wherein the second amplification oligomer is a promoter-primer; and / or Wherein the fourth amplification oligomer is a promoter-primer.

10. The composition, kit or use of claim 9, wherein one or more of the promoter-primers comprises a T7 promoter located 5' to the target hybridizing sequence; and / or wherein one or more promoter-primers comprises a sequence of SEQ ID NO: 8, 9, 10, 11, 12 or 13.

11. A composition or kit as described in any one of claims 1-3, 9 and 10 or a use as described in any one of claims 4-10, wherein one or more of the first, second, third and fourth amplification oligomers comprises a non-nucleotide detectable label.

12. The composition or kit according to any one of claims 1 to 3 and 9 to 11 or the use according to any one of claims 4 to 11, wherein the sequence of the first amplification oligomer is SEQ ID NO: 2; and The composition or kit further comprises an additional amplification oligomer different from the first amplification oligomer, wherein the sequence of the additional amplification oligomer is SEQ ID NO:

3.

13. The composition or kit according to any one of claims 1 to 3 and 9 to 12 or the use according to any one of claims 4 to 12, wherein the sequence of the second amplification oligomer is SEQ ID NO: 21; and / or wherein the sequence of the second amplification oligomer is SEQ ID NO: 14; and / or wherein the sequence of the second amplification oligomer is SEQ ID NO: 22; and / or wherein the second amplification oligomer comprises the sequence of SEQ ID NO: 15; and / or wherein the sequence of the second amplification oligomer is SEQ ID NO: 20; and / or wherein the sequence of the fourth amplification oligomer is SEQ ID NO: 35; and / or wherein the sequence of the fourth amplification oligomer is SEQ ID NO: 34; and / or wherein the sequence of the fourth amplification oligomer is SEQ ID NO: 30; and / or The composition or kit further comprises a fifth amplification oligomer comprising at least 10 consecutive nucleotides of SEQ ID NO: 69 and at least about 14 consecutive nucleotides of a hepatitis B virus sequence or its complement.

14. The composition, kit or use of claim 13, wherein the fifth amplification oligomer comprises at least one or two of SEQ ID NO: 70, 71 or 72; and / or wherein the fifth amplification oligomer comprises at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 consecutive nucleotides of SEQ ID NO: 69; and / or wherein the fifth amplification oligomer comprises the sequence of SEQ ID NO: 69; and / or wherein the fifth amplification oligomer is a promoter-primer; and / or Wherein the fifth amplification oligomer comprises the sequence of SEQ ID NO:

67.

15. The composition or kit of any one of claims 1-3 and 9-14 or the use of any one of claims 4-14, wherein the composition or kit further comprises a sixth amplification oligomer comprising at least 10 consecutive nucleotides of the sequence of SEQ ID NO: 73 and at least about 14 consecutive nucleotides of the hepatitis B virus sequence or its complement.

16. The composition, kit or use of claim 15, wherein the sixth amplification oligomer comprises at least one or two of SEQ ID NO: 74, 75 or 76.

17. The composition, kit or use of claim 15 or 16, wherein the sixth amplification oligomer comprises at least 11, 12, 13, 14, 15, 16, 17, 18 or 19 consecutive nucleotides of SEQ ID NO: 73; and / or wherein the sixth amplification oligomer comprises the sequence of SEQ ID NO: 73; and / or wherein the composition further comprises HBV nucleic acid; and / or wherein the composition or kit further comprises at least one DNA polymerase; and / or Wherein the DNA polymerase is a reverse transcriptase.

18. The composition, kit or use of claim 17, wherein the DNA polymerase is thermophilic.

19. The composition, kit or use of claim 17, wherein the DNA polymerase is mesophilic.

20. The composition or kit of any one of claims 1 to 3 and 9 to 19 or the use of any one of claims 4 to 19, wherein the composition or kit further comprises an RNA polymerase.

21. The composition, kit or use of claim 20, wherein the RNA polymerase is T7 RNA polymerase.

22. The composition or kit of any one of claims 1-3 and 9-21 or the use of any one of claims 4-21, wherein the composition or kit further comprises at least one, at least two or each of Mg2+, a buffer and dNTPs; and / or The composition or kit further comprises rNTPs.

23. A composition or kit as described in any of the preceding claims 1-3 and 9-22 or the use as described in any of claims 4-22, wherein the composition or kit further comprises a first control amplification oligomer and a second control amplification oligomer that do not specifically hybridize to HBV.

24. The composition, kit or use of claim 23, wherein the first control amplification oligomer comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 consecutive nucleotides of the sequence of SEQ ID NO:

77.

25. The composition, kit or use of claim 23 or 24, wherein the second control amplification oligomer comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 consecutive nucleotides of the sequence of SEQ ID NO: 86; and / or wherein the first control amplification oligomer or the second control amplification oligomer is a promoter-primer; and / or wherein the composition or kit further comprises at least one control probe oligomer configured to specifically hybridize to the amplicons produced by the first and the second control amplification oligomers; optionally wherein the control probe oligomer comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 consecutive nucleotides of the sequence of SEQ ID NO:

79.

26. The composition or kit of any one of claims 1-3 and 9-25 or the use of any one of claims 4-25, wherein the probe oligomer comprises the sequence of SEQ ID NO: 29, wherein the sequence of SEQ ID NO: 29 comprises a c9 linker between nucleotides 33 and 34 of SEQ ID NO:

29.

27. A composition or kit as described in any one of claims 1-3 and 9-26 or the use as described in any one of claims 4-26, wherein the composition or kit further comprises at least one probe oligomer configured to specifically hybridize with the amplicons produced by the third and fourth amplification oligomers.

28. The composition or kit of any one of claims 1-3 and 9-27 or the use of any one of claims 4-27, wherein the probe oligomer comprises a non-nucleotide detectable label.

29. The composition, kit or use of claim 28, wherein the probe oligomer further comprises a fluorescent label.

30. The composition, kit or use of claim 29, wherein the probe oligomer further comprises a quencher, and the quencher absorbs fluorescence to a greater extent when the probe is free than when the probe is annealed to a target nucleic acid.

31. The composition, kit or use of claim 30, wherein the fluorescent label is attached to the 5'-end of the probe oligomer and the quencher is attached to the 3'-end of the probe oligomer, or the fluorescent label is attached to the 3'-end of the probe oligomer and the quencher is attached to the 5'-end of the probe oligomer.

32. The composition or kit of any one of claims 1-3 and 9-31 or the use of any one of claims 4-31, wherein the probe oligomer comprises a first self-complementary region at its 5' end and a second self-complementary region at its 3' end.

33. The composition or kit of any one of claims 1, 3 and 9-32 or the use of any one of claims 4-32, wherein the sequence of the target hybridization region of the fourth amplification oligomer is SEQ ID NO: 35.

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