Detection of HIV-1 by nucleic acid amplification
View Patent ↗Nucleic acid sequences and methods for detecting HIV-1 nucleic acid (LTR and pol sequences) in biological samples by detecting amplified nucleic acids are disclosed. Kits comprising nucleic acid oligomers for amplifying HIV-1 nucleic acid present in a biological sample and detecting the amplified nucleic acid are disclosed.
1. A method of detecting HIV-1 nucleic acid in a biological sample, comprising the steps of:
contacting a biological sample containing HIV-1 nucleic acid with at least one capture oligomer comprising an LTR-specific sequence consisting of SEQ ID NO:1 or SEQ ID NO:19 or the LTR-specific sequence contained in SEQ ID NO:2 or SEQ ID NO:20 that hybridizes specifically to a target region in LTR sequences of HIV-1 nucleic acid and at least one capture oligomer comprising a pol-specific sequence consisting of SEQ ID NO:3 or SEQ ID NO:5 or the pol-specific sequence contained in SEQ ID NO:4 or SEQ ID NO:6 that hybridizes specifically to a target region in pol sequences of HIV-1 nucleic acid, thus forming a capture oligomer:HIV-1 nucleic acid complex;
separating the capture oligomer:HIV-1 nucleic acid complex from the biological sample;
amplifying LTR sequences, or a cDNA made therefrom, by using at least two amplification oligomers, wherein a first amplification oligomer is selected from the group consisting of SEQ ID NO:7 optionally with a promoter sequence joined to its 5′ end, SEQ ID NO:8, SEQ ID NO:29 optionally with a promoter sequence joined to its 5′ end, SEQ ID NO:30, SEQ ID NO:31 optionally with a promoter sequence joined to its 5′ end, SEQ ID NO:32, SEQ ID NO:33 optionally with a promoter sequence joined to its 5′ end, and SEQ ID NO:34, and wherein a second amplification oligomer is selected from the group consisting of SEQ ID NO:9, SEQ ID NO:35, and SEQ ID NO:36, and a nucleic acid polymerase in vitro to produce an amplified product of LTR sequences; and
detecting the amplified product of LTR sequences using a labeled detection probe that hybridizes specifically with LTR sequence in the amplified product, thereby indicating presence of the HIV-1 nucleic acid in the biological sample.
2. The method of claim 1 , wherein the contacting step uses a capture oligomer that hybridizes specifically to a target region in LTR sequences of HIV-1 nucleic acid complementary to SEQ ID NO:1 or complementary to LTR-specific sequence contained in SEQ ID NO:2.
3. The method of claim 1 , wherein the contacting step uses a capture oligomer that hybridizes specifically to a target region in LTR sequences of HIV-1 nucleic acid complementary to SEQ ID NO:19 or complementary to LTR-specific sequence contained in SEQ ID NO:20.
4. The method of claim 1 , wherein the amplifying step uses at least two amplification oligomers, wherein the first amplification oligomer is a promoter primer consisting essentially of SEQ ID NO:8, and the second amplification oligomer is a primer consisting essentially of SEQ ID NO:9, SEQ ID NO:35 or SEQ ID NO:36.
5. The method of claim 1 , wherein the amplifying step uses any of the following combinations of amplification oligomers:
a promoter-primer consisting essentially of SEQ ID NO:8 with the primer consisting essentially of SEQ ID NO:9;
a promoter-primer consisting essentially of SEQ ID NO:8 with the primer consisting essentially of SEQ ID NO:35;
a promoter-primer consisting essentially of SEQ ID NO:8 with the primer consisting essentially of SEQ ID NO:36;
a promoter-primer consisting essentially of SEQ ID NO:30 with the primer consisting essentially of SEQ ID NO:9;
a promoter-primer consisting essentially of SEQ ID NO:32 with the primer consisting essentially of SEQ ID NO:9; and
a promoter-primer consisting essentially of SEQ ID NO:34 with the primer consisting essentially of SEQ ID NO:9.
6. The method of claim 1 , wherein the amplifying step further uses at least two amplification oligomers that bind specifically to HIV-1 pol sequences or to sequences complementary to HIV-1 pol sequences.
7. The method of claim 6 , wherein the amplifying step uses a mixture of amplification oligomers that bind specifically to HIV-1 pol sequences consisting essentially of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, and SEQ ID NO:15.
8. The method of claim 1 , wherein the detecting step uses at least one labeled detection probe having an LTR-specific sequence consisting essentially of SEQ ID NO:16 or SEQ ID NO:41.
9. The method of claim 1 , wherein the detecting step uses a mixture of at least two labeled detection probes wherein a first probe consists essentially of SEQ ID NO:16 and a second probe consists essentially of SEQ ID NO:41.
10. The method of claim 1 , wherein the detecting step uses at least one labeled detection probe that includes at least one 2′-methoxy backbone linkage.
11. A kit comprising a combination of at least two capture oligomers, wherein a first capture oligomer hybridizes specifically to a target region in LTR sequences of HIV-1 nucleic acid and a second capture oligomer hybridizes specifically to a target region in pol sequences of HIV-1 nucleic acid, and at least two amplification oligomers to produce an amplified product of LTR sequences of HIV-1 nucleic acid.
12. The kit of claim 11 wherein the first capture oligomer is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:19, SEQ ID NO:2 and SEQ ID NO:20 that hybridizes specifically to a target region in LTR sequences, and wherein the second capture oligomer is selected from the group consisting of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:4, and SEQ ID NO:6 that hybridizes specifically to a target region in pol sequences.
13. The kit of claim 11 wherein the at least two amplification oligomers to produce an amplified product of LTR sequences of HIV-1 nucleic acid comprise at least one first amplification oligomer selected from the group consisting of SEQ ID NO:7 optionally with a promoter sequence joined to its 5′ end, SEQ ID NO:8, SEQ ID NO:29 optionally with a promoter sequence joined to its 5′ end, SEQ ID NO:30, SEQ ID NO:31 optionally with a promoter sequence joined to its 5′ end, SEQ ID NO:32, SEQ ID NO:33 optionally with a promoter sequence joined to its 5′ end, and SEQ ID NO:34, and at least one second amplification oligomer is selected from the group consisting of SEQ ID NO:9, SEQ ID NO:35, and SEQ ID NO:36.
14. The kit of claim 11 further comprising at least one labeled detection probe having an LTR-specific sequence consisting essentially of SEQ ID NO:16 or SEQ ID NO:41.
15. A composition comprising a combination of at least two capture oligomers, wherein a first capture oligomer hybridizes specifically to a target region in LTR sequences of HIV-1 nucleic acid and a second capture oligomer hybridizes specifically to a target region in pol sequences of HIV-1 nucleic acid, and at least two amplification oligomers, wherein a first amplification oligomer is selected from the group consisting of SEQ ID NO:7 optionally with a promoter sequence joined to its 5′ end, SEQ ID NO:8, SEQ ID NO:29 optionally with a promoter sequence joined to its 5′ end, SEQ ID NO:30, SEQ ID NO:31 optionally with a promoter sequence joined to its 5′ end, SEQ ID NO:32, SEQ ID NO:33 optionally with a promoter sequence joined to its 5′ end, and SEQ ID NO:34, and wherein a second amplification oligomer is selected from the group consisting of SEQ ID NO:9, SEQ ID NO:35, and SEQ ID NO:36.
16. The composition of claim 15 wherein the promoter sequence is a T7 RNA polymerase promoter sequence.
17. The composition of claim 15 wherein the first capture oligomer is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:19, SEQ ID NO:2 and SEQ ID NO:20 that hybridizes specifically to a target region in LTR sequences, and wherein the second capture oligomer is selected from the group consisting of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:4, and SEQ ID NO:6 that hybridizes specifically to a target region in pol sequences.
18. The composition of claim 15 wherein an oligomer sequence is linked by a backbone that includes at least one 2′-methoxy RNA group, at least one 2′ fluoro-substituted RNA group, at least one peptide nucleic acid linkage, at least one phosphorothioate linkage, at least one methylphosphonate linkage or any combination thereof.
19. The composition of claim 15 wherein the oligomer or an oligomer in the mixture of oligomers comprises at least one 2′-methoxy RNA group in the backbone.
20. The composition of claim 15 further comprising an oligomer consisting essentially of SEQ ID NO:16 or SEQ ID NO:41 linked to a detectable label.