Amplification of HIV-1 gag sequences for detection of sequences associated with drug-resistance mutations
View Patent ↗Sequences of nucleic acid oligonucleotides for amplifying different portions of gag and pol genes of HIV-1 and for detecting such amplified nucleic acid sequences are disclosed. Methods of amplifying and detecting HIV-1 nucleic acid in a biological sample using the amplification oligonucleotides specific for gag and pol target sequences are disclosed.
1. A mixture of nucleic acid oligomers for amplifying a nucleotide sequence of HIV-1 in a gag region referred to as a first gag sequence, comprising a sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:17, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:46, SEQ ID NO:47, and SEQ ID NO:59.
2. A nucleic acid oligomer according to claim 1 , wherein the oligomer nucleic acid backbone comprises one or more 2′-methoxy linkages, peptide nucleic acid linkages, phosphorothioate linkages, methylphosphonate linkages or any combination of these linkages.
3. A nucleic acid oligomer according to claim 1 , wherein the oligomer is a promoter-primer comprising a sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:33, and SEQ ID NO:35, wherein a 5′ portion of the sequence includes a promoter sequence for T7 RNA polymerase.
4. A mixture of nucleic acid oligomers according to claim 1 , wherein the mixture further comprises oligomers for amplifying a second gag sequence and having a nucleotide sequence selected from the group consisting of SEQ ID NO:18, and SEQ ID NO:60.
5. A mixture of nucleic acid oligomers according to claim 1 , wherein the mixture further comprises oligomers for amplifying a Protease sequence and having a nucleotide sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:19, SEQ ID NO:61, and SEQ ID NO:62.
6. A mixture of nucleic acid oligomers according to claim 1 , wherein the mixture further comprises oligomers for amplifying a first reverse transcriptase (RI) sequence and having a nucleotide sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:20, SEQ ID NO:63, SEQ ID NO:64 and SEQ ID NO:65.
7. A mixture of nucleic acid oligomers according to claim 1 , wherein the mixture further comprises oligomers for amplifying a second RT sequence and having a nucleotide sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:15, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:21, and SEQ ID NO:66.
8. A mixture of nucleic acid oligomers according to claim 1 , wherein the mixture further comprises oligomers for amplifying a third RI sequence and having a nucleotide sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:16, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:22, SEQ ID NO:67, and SEQ ID NO:68.
9. A mixture of nucleic acid oligomers according to claim 1 , wherein the mixture further comprises a labeled oligonucleotide that specifically hybridizes to an HIV-1 sequence derived from the first gag sequence, having a base sequence selected from the group consisting of SEQ ID NO:23 and SEQ ID NO:24, and a label that results in a detectable signal.
10. A labeled oligonucleotide according to claim 9 , wherein the oligonucleotide includes in its nucleic acid backbone one or more 2′-methoxy linkages, peptide nucleic acid linkages, phosphorothioate linkages, methylphosphonate linkages or any combination of these linkages.
11. A labeled oligonucleotide according to claim 9 , wherein the label is a compound that produces a luminescent signal that can be detected in a homogeneous detection system.
12. A labeled oligonucleotide according to claim 9 , wherein the label is an acridinium ester (AE) compound.
13. A mixture of nucleic acid oliogmers according to claim 1 , further comprising a labeled oligonucleotide that hybridizes to an HIV-1 sequence derived from pc/sequences and has a base sequence selected from the group consisting of: SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29, and a labeled oligonucleotide that hybridizes to an HIV-1 sequence derived from a second gag sequence and has the base sequence of SEQ ID NO:25.
14. A method of detecting HIV-1 in a biological sample, comprising the steps of:
providing a biological sample containing HIV-1 nucleic acid;
mixing the sample with two or more amplification oligomers that specifically amplify at least one HIV-1 target sequence contained within a first gag sequence under conditions that allow amplification of nucleic acid, wherein the amplification oligomers have sequences selected from the group consisting of:
SEQ ID NO:5, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:17, and SEQ ID NO:59 to amplify a first gag sequence;
amplifying the target sequence in the first gag sequence to produce an amplified nucleic acid product; and
detecting the presence of the amplified nucleic acid product.
15. The method of claim 14 , wherein the amplifying step uses a transcription-mediated amplification method which is conducted in substantially isothermal conditions.
16. The method of claim 14 , wherein the detecting step uses:
a labeled oligomer having the sequence of SEQ ID NO:23, SEQ ID NO:24, or a mixture of these oligomers, to hybridize specifically to the amplified nucleic acid product of the first gag sequence.
17. The method of claim 14 , wherein the detecting step detects hybridization of the amplified nucleic acid to an array of nucleic acid probes.
18. The method of claim 14 , further comprising the step of contacting the sample containing HIV-1 nucleic acid with at least one capture oligomer having a sequence that hybridizes specifically to the HIV-1 nucleic acid, thus forming a hybridization complex that includes the HIV-1 nucleic acid and separating the hybridization complex from other sample components.
19. The method of claim 14 , wherein the detecting step detects hybridization of the amplified nucleic acid product to a labeled oligonucleotide that is labeled with a compound that produces a luminescent signal that can be detected in a homogeneous detection system.
20. The method of claim 14 , wherein at least one amplification oligomer includes a nucleic acid backbone that comprises one or more 2′-methoxy linkages.
21. The method of claim 14 , wherein the mixing step further includes mixing two or more amplification oligomers that specifically amplify at least one HIV-1 target sequence contained within a HIV-1 pol sequence, wherein the amplifying step further amplifies a pol target sequence to produce an amplified nucleic acid product of the pol sequence, and wherein the detecting step detects the amplified nucleic acid product of the pol sequence.
22. The method of claim 21 , wherein the detecting step detects hybridization of the amplified nucleic acid product of the pol sequence to at least one nucleic acid probe specific for the pol sequence.
23. The method of claim 14 , wherein the mixing step further includes mixing two or more amplification oligomers that specifically amplify at least one HIV-1 target sequence contained within a HIV-1 second gag sequence, wherein the amplifying step further amplifies a second gag target sequence to produce an amplified nucleic acid product of the second gag sequence, and wherein the detecting step detects the amplified nucleic acid product of the second gag sequence.
24. The method of claim 23 , wherein the detecting step detects hybridization of the amplified nucleic acid product of the second gag sequence to at least one nucleic acid probe specific for the second gag sequence.