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 a base sequence that hybridizes specifically to a target region in LTR or 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:29, SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32, and wherein a second amplification oligomer is selected from the group consisting of SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:38, and a nucleic acid polymerase in vitro to produce an amplified product; and
detecting the amplified product using a labeled detection probe that hybridizes specifically with 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 SEQ ID NO:57, or complementary to LTR-specific contained in SEQ ID NO:2 or SEQ ID NO:45.
3. The method of claim 1 , wherein the contacting step further comprises using a capture oligomer that hybridizes specifically to a target region in pol sequences of HIV-1 nucleic acid.
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 having the sequence of SEQ ID NO:30 or SEQ ID NO:32, and the second amplification oligomer is a primer having the sequence of SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, or SEQ ID NO:38.
5. The method of claim 4 , wherein the amplifying step uses any of the following combinations of promoter-primers and primers:
the promoter-primer consisting essentially of SEQ ID NO:30 with the primer consisting essentially of SEQ ID NO:35;
the promoter-primer consisting essentially of SEQ ID NO:30 with the primer consisting essentially of SEQ ID NO:36;
the promoter-primer consisting essentially of SEQ ID NO:30 with the primer consisting essentially of SEQ ID NO:37;
the promoter-primer consisting essentially of SEQ ID NO:30 with the primer consisting essentially of SEQ ID NO:38;
the promoter-primer consisting essentially of SEQ ID NO:32 with the primer consisting essentially of SEQ ID NO:35;
the promoter-primer consisting essentially of SEQ ID NO:32 with the primer consisting essentially of SEQ ID NO:36;
the promoter-primer consisting essentially of SEQ ID NO:32 with the primer consisting essentially of SEQ ID NO:37; and
the promoter-primer consisting essentially of SEQ ID NO:32 with the primer consisting essentially of SEQ ID NO:38.
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 at least two amplification oligomers for amplifying pol sequences selected from the group consisting of: SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:42, SEQ ID NO:43, and SEQ ID NO:44.
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, SEQ ID NO:39, SEQ ID NO:40, or SEQ ID NO:41.
9. The method of claim 1 , wherein the detecting step uses a combination of at least two labeled detection probes made up of a first probe having a sequence consisting essentially of SEQ ID NO:16 and a second probe having a sequence consisting essentially of SEQ ID NO:41.
10. The method of claim 1 , wherein the detecting step uses a combination of at least two labeled detection probes made up of a first probe sequence that is about 20 bases containing a sequence of 18 bases contained in SEQ ID NO:39, and a second probe sequence that is about 20 bases containing a sequence of 14 bases contained in SEQ ID NO:40.
11. The method of claim 1 , wherein the detecting step uses a combination of at least two labeled detection probes made up of at least one probe that has a sequence of about 20 bases consisting essentially of SEQ ID NO:16 and at least one probe that has a pol-specific sequence.
12. 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.
13. A kit comprising a combination of at least two oligomers, wherein LTR-specific oligomers contained in the kit are selected from the group consisting of SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:38.
14. The kit of claim 13 , further comprising at least one oligomer having an LTR-specific sequence consisting essentially of SEQ ID NO:16, SEQ ID NO:39, SEQ ID NO:40, or SEQ ID NO:41.
15. A composition comprising a combination of at least two oligomers, wherein a first oligomer is selected from the group consisting of:
SEQ ID NO:29, optionally with a promoter sequence covalently attached to the 5′ end of SEQ ID NO:29;
SEQ ID NO:31, optionally with a promoter sequence covalently attached to the 5′ end of SEQ ID NO:31;
SEQ ID NO:30; and
SEQ ID NO:32,
and wherein a second oligomer is selected from the group consisting of SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:38.
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 composition further comprises an oligomer of SEQ ID NO:16, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, or a mixture of oligomers of SEQ ID NO:16 and SEQ ID NO:41.
18. The composition of claim 15 , wherein an oligomer base 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 17 , 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 17 , wherein the oligomer or an oligomer in the mixture of oligomers is linked to a detectable label.