Compositions and methods for detecting hepatitis B virus
View Patent ↗Compositions, methods and kits for detecting viral nucleic acids. Targets that can be detected in accordance with the invention include HBV and/or HIV-1 and/or HCV nucleic acids. Particularly described are oligonucleotides that are useful as hybridization probes and amplification primers that facilitate detection of very low levels of HBV nucleic acids.
1. A method of detecting HBV nucleic acid contained in a test sample comprising nucleic acids, comprising the steps of:
(a) contacting the test sample with at least two amplification oligomers, wherein a first amplification oligomer comprises a target-hybridizing region having a nucleotide sequence consisting of SEQ ID NO:26;
(b) amplifying any HBV nucleic acid that may be present in the test sample in an in vitro nucleic acid amplification reaction, whereby there are synthesized HBV amplicons if the test sample contained HBV nucleic acid; and
(c) detecting the HBV amplicons, if present, with at least one hybridization assay probe, thereby detecting HBV nucleic acid contained in the test sample, wherein the at least one hybridization assay probe is at least 17 contiguous nucleotides contained within a sequence consisting of SEQ ID NO:67 or the complement thereof, allowing for the presence of RNA equivalents and nucleotide analogs.
2. The method of claim 1 , wherein the detecting step comprises detecting said HBV amplicons after the in vitro nucleic acid amplification reaction has terminated.
3. The method of claim 1 , wherein the detecting step comprises detecting said HBV amplicons while the in vitro nucleic acid amplification reaction is occurring.
4. The method of claim 3 , wherein the in vitro nucleic acid amplification reaction is an isothermal nucleic acid amplification reaction.
5. The method of claim 4 , wherein the in vitro nucleic acid amplification reaction is a multiplex nucleic acid amplification reaction that amplifies HIV-1 nucleic acid and HCV nucleic acid in addition to HBV nucleic acid.
6. The method of claim 1 , wherein the detecting step comprises detecting HBV genotype G amplicons.
7. The method of claim 1 , wherein the first amplification oligomer comprises a nucleotide sequence consisting of SEQ ID NO:43.
8. The method of claim 7 , wherein a second amplification oligomer comprises a nucleotide sequence consisting of SEQ ID NO:11.
9. The method of claim 1 , wherein the at least one hybridization assay probe nucleotide sequence-consists of a sequence-selected form the group consisting of SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:65, the RNA equivalent of SEQ ID NO:52, and the RNA equivalent of SEQ ID NO:58.
10. The method of claim 9 , wherein the at least one hybridization assay probe further comprises a detectable label selected from the group consisting of: luminescent compounds, bioluminescent compounds, phosphorescent compounds, chemiluminescent compounds, and acridinium ester compounds.
11. The method of claim 1 , wherein the at least one hybridization assay probe is 17-23 contiguous nucleotides contained within a sequence consisting of SEQ ID NO:95 or the complement thereof, allowing for the presence of RNA equivalents and nucleotide analogs.
12. The method of claim 11 , wherein the at least one hybridization assay probe comprises a contiguous RNA nucleotide sequence selected form the group consisting of SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:65, the RNA equivalent of SEQ ID NO:52, and the RNA equivalent of SEQ ID NO:58.
13. The method of claim 12 , wherein the at least one hybridization assay probe further comprises a detectable label selected from the group consisting of: luminescent compounds, bioluminescent compounds, phosphorescent compounds, chemiluminescent compounds, and acridinium ester compounds.
14. The method of claim 1 , wherein the at least one hybridization assay probe further comprises a detectable label selected from the group consisting of: radioisotopes, enzymes, haptens, chromophores, dyes, luminescent compounds, bioluminescent compounds, phosphorescent compounds, chemiluminescent compounds, fluorescent compounds, and acridinium ester compounds.
15. The method of claim 1 , wherein the at least one hybridization probe is at least 17 contiguous RNA nucleotides.
16. The method of claim 15 , wherein at least one of the RNA nucleotides contains a 2′-O-Methyl modified nucleobase.
17. The method of claim 1 , wherein the at least one hybridization assay probe is a combination of DNA and RNA.
18. The method of claim 17 , wherein at least one of the RNA nucleotides contains a 2′-O-Methyl modified nucleobase.
19. The method of claim 1 , wherein the at least one hybridization assay probe contains a sequence consisting of nucleotides 37-54 SEQ ID NO:67 or the complement thereof, allowing for the presence of RNA equivalents and nucleotide analogs.
20. The method of claim 1 , wherein the at least one hybridization assay probe is 17-19 contiguous nucleotides contained within a sequence consisting of SEQ ID NO:95 or the complement thereof, allowing for the presence of RNA equivalents and nucleotide analogs.
21. The method of claim 20 , wherein the at least one hybridization assay probe comprises a contiguous RNA nucleotide sequence-selected form the group consisting of SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:65, the RNA equivalent of SEQ ID NO:52, and the RNA equivalent of SEQ ID NO:58.
22. The method of claim 21 , wherein the at least one hybridization assay probe further comprises a detectable label selected from the group consisting of: luminescent compounds, bioluminescent compounds, phosphorescent compounds, chemiluminescent compounds, and acridinium ester compounds.