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:
providing a biological sample containing HIV-1 nucleic acid;
contacting the biological sample with at least one capture oligomer comprising a base sequence that hybridizes specifically to a target region in LTR or pot 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 pol sequences, or a cDNA made therefrom, by using at least two amplification oligomers selected from the group consisting of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:42, SEQ ID NO:43, or SEQ ID NO:44 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 pal sequences of HIV-1 nucleic acid complementary to SEQ ID NO:3 or SEQ ID NO:5, or a combination of oligomers that hybridize specifically to target regions in pol sequences of HIV-1 nucleic acid complementary to SEQ ID NO:3 and SEQ ID NO:5.
3. The method of claim 2 , wherein the contacting step further comprises using a capture oligomer that hybridizes specifically to a target region in LTR sequences of HIV-1 nucleic acid.
4. The method of claim 1 , wherein the amplifying step uses at least two amplification cligomers, wherein a first amplification oligomer is a promoter primer having the sequence of SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:43, or SEQ ID NO:44, and a second amplification oligomer is a primer having the sequence of SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:42.
5. The method of claim 4 , wherein the amplifying step uses any of the following combinations of promoter-primers and primers;
promoter-primers consisting essentially of SEQ ID NO:13 and SEQ ID NO:15, with primers consisting essentially of SEQ ID NO:10 and SEQ ID NO:11;
promoter-primers consisting essentially of SEQ ID NO:13 and SEQ ID NO:15, with primers consisting essentially of SEQ ID NO:11 and SEQ ID NO:42; or
promoter-primers consisting essentially of SEQ ID NO:43 and SEQ ID NO:15, with primers consisting essentially of SEQ ID NO:10 and SEQ ID NO:11.
6. The method of claim 1 , wherein the amplifying step further uses at least two amplification oligomers that bind specifically to LTR sequences or to sequences complementary to LTR sequences.
7. The method of claim 6 , wherein the amplifying step uses at least two amplification ollgomers for amplifying LTR sequences selected from the group consisting of: SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO;21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO;34, SEQ ID NO:35, SEQ ID NO;36, SEQ ID NO;37, and SEQ ID NO:38.
8. The method of claim 1 , wherein the detecting step uses at least one labeled detection probe having a pol-specific sequence consisting essentially of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56.
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 sequence that is about 22 to about 30 bases consisting essentially of SEQ ID NO:17, and a second probe sequence that is about 17 to about 20 bases consisting essentially of SEQ ID NO:18.
10. 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 pol-specific sequence of about 22 to about 30 bases consisting essentially of SEQ ID NO:17 or about 17 to about 20 bases consisting essentially of SEQ ID NO:18, and at least one probe that has a LTR-specific sequence.
11. 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.
12. A kit comprising a combination of oligomers, wherein oligomers contained in the kit have sequences consisting essentially of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, and SEQ ID NO:15.
13. The kit of claim 12 , further comprising oligomers having base sequences consisting essentially of SEQ ID NO:17 and SEQ ID NO:18.
14. A composition comprising a combination of at least two oligomers selected from the group consisting of:
SEQ ID NO:12, optionally with a promoter sequence covalently attached to the 5′ end of SEQ ID NO:12;
SEQ ID NO:14, optionally with a promoter sequence covalently attached to the 5′ end of SEQ ID NO:14:
SEQ ID NO:10;
SEQ ID NO:11; and
SEQ ID NO:42.
15. The composition of claim 14 , wherein the promoter sequence is a T7 RNA polymerase promoter sequence.
16. The composition of claim 14 , wherein the composition further comprises an oligomer of SEQ ID NO:17, SEQ ID NO:18, or a mixture of oligomers of SEQ ID NO:17 and SEQ ID NO:18.
17. The composition of claim 14 , 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.
18. The composition of claim 14 , 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 16 , wherein the oligomer comprises at least one 2′-methoxy RNA group in the backbone.
20. The composition of claim 16 , wherein the oligomer is linked to a detectable label.